Binding proteins and methods of use thereof

ABSTRACT

The present disclosure provides binding proteins, such as antibodies, that bind to a GDNF Family Receptor Alpha Like (GFRAL) protein, including human GFRAL protein, and methods of their use.

This application is a divisional of U.S. application Ser. No. 15/449,839, filed Mar. 3, 2017, which claims the benefit of U.S. Provisional Application No. 62/316,516, filed Mar. 31, 2016, the entire contents of each of which are incorporated herein by reference.

FIELD

The present disclosure relates generally to binding proteins, such as antibodies, that bind to a GDNF Family Receptor Alpha Like (GFRAL) protein, including human GFRAL protein, and methods of their use.

BACKGROUND

Growth differentiation factor 15 (GDF15) is a protein belonging to the transforming growth factor beta (TGF-β) superfamily. GDF15 is also known as TGF-PL, MIC-1, PDF, PLAB, NAG-1, and PTGFB. GDF15 mRNA is reported to be most abundant in the liver, with lower levels seen in some other tissues. Its expression in liver can be significantly up-regulated in injury of organs such as liver, kidney, heart and lung.

GDF15 is reported to play a role in regulating inflammatory and apoptotic pathways in injured tissues and during disease processes. It has been reported that GDF15 is a mediator of cachexia in various diseases. However, cachexia is a complex and incompletely understood syndrome. In addition, at least some tumors over-express and secrete GDF15, and elevated serum GDF15 levels have been associated with various cancers. GDF-15 has been described as a negative regulator of macrophage activation by suppressing the release of TNF-α, IL-1, IL-2 and MCS-F, thus inhibiting the positive feedback of local inflammatory signaling similar to the effects of TGF-β. Monoclonal antibodies against GDF15 have been disclosed as potential therapeutic agents for the treatment of cachexia and of cancer. The receptor for GDF15 is unknown.

There is a significant unmet need for therapeutic agents effective to treat weight loss associated with a number of diseases and conditions, including wasting diseases such as cachexia or sarcopenia and inflammatory conditions such as systemic inflammation or an acute inflammatory response. There is also a significant unmet need for therapeutic agents effective to treat chronic diseases, including cancer, chronic renal disease, chronic obstructive pulmonary disease, AIDS, tuberculosis, chronic inflammatory disease, systemic inflammation, and muscle wasting diseases, including in which involuntary body weight loss and/or muscle mass loss is involved.

SUMMARY

The present disclosure provides proteins that bind to a GDNF Family Receptor Alpha Like (GFRAL) protein, including binding proteins such as antibodies that bind to a GFRAL protein. Such binding proteins including antibodies, may bind to a GFRAL polypeptide, a GFRAL fragment and/or a GFRAL epitope. Such binding proteins, including antibodies, may be antagonists (e.g., inhibit binding of a GDF15 protein to a GFRAL protein, inhibit binding of a RET protein to a GFRAL protein, inhibit a GDF15 protein induced signaling, and/or inhibit formation of a GDF15/GFRAL or a GDF15/GFRAL/RET receptor complex).

The present disclosure also provides binding proteins, including antibodies (e.g., monoclonal antibodies) or fragments thereof, that (i) bind to a GFRAL protein, (ii) inhibit binding of a GDF15 protein to a GFRAL protein, and/or (iii) inhibit binding of a RET protein to a GFRAL protein.

In some embodiments, the anti-GFRAL antibodies are humanized antibodies that bind to a GFRAL polypeptide, a GFRAL fragment, or a GFRAL epitope. In certain embodiments, an anti-GFRAL antibody comprises a VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a monoclonal antibody designated 1C1, 3P10, 12A3, 5F12, 5A20, 8D8, 17J16, 25M22, 2B8, 22N5, 2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9, 24G2, 6G9, 2B11, 1A3, P1B6, P1H8, or P8G4 as described herein, or a humanized variant thereof. In certain embodiments, an anti-GFRAL antibody can further comprise a VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3, and/or VL FR4 of a human immunoglobulin amino acid sequence or a variant thereof.

In some embodiments, a binding protein (e.g., an anti-GFRAL antibody) comprises six CDRs or less than six CDRs. In some embodiments, a binding protein (e.g., an anti-GFRAL antibody) comprises one, two, three, four, five, or six CDRs selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3. In some embodiments, a binding protein (e.g., an anti-GFRAL antibody) comprises one, two, three, four, five, or six CDRs selected from VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a monoclonal antibody designated as 1C1, 3P10, 12A3, 5F12, 5A20, 8D8, 17J16, 25M22, 2B8, 22N5, 2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9, 24G2, 6G9, 2B11, 1A3, P1B6, P1H8, or P8G4 as described herein, or a humanized variant thereof. In some embodiments, a binding protein (e.g., an anti-GFRAL antibody) further comprises a scaffold region or framework region, including a VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3, and/or VL FR4 of a human immunoglobulin amino acid sequence or a variant thereof.

In some embodiments, the antibody is a humanized antibody, a monoclonal antibody, a recombinant antibody, an antigen binding fragment or any combination thereof. In some embodiments, the antibody is a humanized monoclonal antibody, or antigen binding fragment thereof, that binds to a GFRAL polypeptide (e.g., a cell surface-expressed or soluble GFRAL), a GFRAL fragment, or a GFRAL epitope.

The present disclosure also provides binding proteins such as anti-GFRAL antibodies (i) that competitively block (e.g., in a dose-dependent manner) an anti-GFRAL antibody provided herein from binding to a GFRAL polypeptide (e.g., a cell surface-expressed or soluble GFRAL), a GFRAL fragment, or a GFRAL epitope and/or (ii) that bind to a GFRAL epitope that is bound by an anti-GFRAL antibody provided herein. In other embodiments, the binding proteins such as anti-GFRAL antibody competitively block (e.g., in a dose-dependent manner) monoclonal antibody 25M22, 3P10, 8D8 or 5F12 described herein or a humanized variant thereof from binding to a GFRAL polypeptide (e.g., a cell surface-expressed or soluble GFRAL protein), a GFRAL fragment, or a GFRAL epitope. In other embodiments, the binding proteins such as anti-GFRAL antibody bind to a GFRAL epitope that is bound (e.g., recognized) by monoclonal antibody 25M22, 3P10, 8D8 or 5F12 described herein or a humanized variant thereof.

The present disclosure also provides binding proteins, including antibodies or fragments thereof, that (i) bind to an epitope of a GFRAL protein recognized by an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3, 7, 11, or 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 4, 8, 12, or 16, respectively; or (ii) compete for the binding to a GFRAL protein with an antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 3, 7, 11, or 15 and a light chain variable region having the amino acid sequence of SEQ ID NO: 4, 8, 12, or 16, respectively. In some embodiments, binding proteins, including antibodies or fragments thereof, are provided herein that bind to a region, including an epitope, of one or more amino acids of a GFRAL protein (e.g., a human GFRAL protein). In some embodiments, binding proteins, including antibodies or fragments thereof, bind to a region of a GFRAL protein (e.g., one or more amino acid residues of an extracellular domain) including, for example, those that bind to: (i) domain 1 of a GFRAL protein (e.g., amino acid residues Q20 to S130 of SEQ ID NO: 1797); (ii) domain 2 of a GFRAL protein (e.g., amino acid residues C131 to C210 of SEQ ID NO: 1797); (iii) domain 3 of a GFRAL protein (e.g., amino acid residues C220 to C316 of SEQ ID NO: 1797); or (iv) an extracellular domain of a GFRAL protein (e.g., amino acid residues Q20 to E351 of SEQ ID NO: 1797).

In some embodiments, binding proteins, including antibodies or fragments thereof, are provided herein that bind to a specific epitope (e.g., one or more amino acid residues) of a GFRAL protein, including, for example, those that bind to: (i) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues SER156, GLN147, LEU148, ALA149, SER150, TYR151, LEU152, LYS153, ALA154, CYS155, PHE174, TYR175, GLU136, ALA137, CYS138, VAL139, GLY140, ASP141, VAL142, VAL143, CYS144, ASN145, ALA146, LEU186, CYS189, CYS191, ALA192, GLN193, SER194, ASP195, ILE196, PRO197, CYS198, GLN199, GLN200, SER201, LYS202, GLU203, ALA204, LEU205, HIS206, SER207, SER130, CYS131, LEU132, GLU133, VAL134, orALA135 of a GFRAL protein (SEQ ID NO: 1797); (ii) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues LEU132, GLU133, VAL134, ALA135, GLU136, ALA137, CYS138, VAL139, GLY140, ASP141, VAL142, VAL143, CYS144, ASN145, ALA146, GLN147, LEU148, ALA149, SER150, TYR151, PHE174, TYR175, ALA169, ALA170, ILE171, ARG172, PHE173, GLN176, ASN177, ILE178, PRO179, PHE180, ASN181, ILE182, ALA183, GLN184, MET185, LEU186, ALA187, PHE188, or CYS189 of SEQ ID NO: 1797; (iii) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues LEU164, LYS208, VAL212, ASN213, MET214, VAL215, PRO216, PRO217, PRO218, THR219, CYS220, LEU221, VAL223, TRP245, LEU267, CYS269, GLN28, VAL289, GLN290, CYS291, THR292, CYS293, ARG294, THR295, ILE296, THR297, GLN298, SER299, GLU300, GLU301, SER302, LEU303, CYS304, LYS305, ILE306, PHE307, GLN308, HIS309, MET310, LEU311, HIS312, ARG313, LYS314, SER315, CYS316, or PHE317 of SEQ ID NO: 1797; (iv) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues CYS233, ARG234, ARG235, HIS236, TYR237, ARG238, THR239, PHE240, GLN241, SER242, LYS243, CYS244, TRP245, GLN246, ARG247, VAL248, THR249, ARG250, LYS251, CYS252, HIS253, GLU254, ASP255, GLU256, ASN257, CYS258, ILE259, SER260, THR261, LEU262, SER263, LYS264, ASP266, LEU267, THR268, SER272, ASP274, CYS275, ALA278, CYS269, SER270, SER302, LEU303, ILE306, HIS309, LEU311, MET310, SER315, or CYS316 of SEQ ID NO: 1797; (v) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues GLY140, LEU148, ALA149, ALA146, VAL142, ASN145, VAL139, ALA135, GLU136, LEU152, LEU132, SER201, ALA204, LEU205, LYS153, ILE196, PRO197, or GLN200 of SEQ ID NO: 1797; (vi) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues GLU136, ALA137, VAL139, GLY140, ASP141, VAL142, VAL143, CYS144, ASN145, ALA146, GLN147, PHE173, ASN177, ILE178, PRO179, ASN181, ILE182, or MET185 of SEQ ID NO: 1797; (vii) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues GLN298 or GLU301 of SEQ ID NO: 1797; or (viii) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues ARG234, ARG238, GLN241, SER242, LYS243, TRP245, GLN246, THR249, ARG250, LYS251, CYS252, HIS253, ASP255, ASN257, CYS258, SER260, THR261, or LEU262 of SEQ ID NO: 1797. Such antibodies provided above can, in some embodiments, inhibit GDF15-induced signaling and/or signaling or activation of a GFRAL/GDF15 or RET/GFRAL/GDF15 receptor complex, for example, in a cell that expresses a GFRAL protein. Additionally, in some embodiments, the antibody is a monoclonal antibody, for example, a humanized, human or chimeric antibody.

In some embodiments, the binding proteins such as anti-GFRAL antibodies provided herein are conjugated or recombinantly linked to a diagnostic agent, a detectable agent (e.g., a radioisotope, an enzyme, a fluorescent compound, a bioluminescent compound or a chemiluminescent compound). In some embodiments, the binding proteins such as anti-GFRAL antibodies provided herein are used (e.g., administered) with a therapeutic agent. In some aspects, the therapeutic agent is a drug, including one or more drugs such as an inhibitor of Activin-A, an inhibitor of ActRIIB, an inhibitor of IL-6 or an inhibitor of IL-6R, a ghrelin, a ghrelin mimetic or a GHS-RIa agonist, a SARM, a TNFα inhibitor, an IL-Ia inhibitor, a myostatin inhibitor, a beta-blocker, a melanocortin peptide inhibitor, a melanocortin receptor inhibitor, or an anti-cancer agent.

In certain embodiments, compositions are provided comprising a binding protein such as an anti-GFRAL antibody described herein. Also provided herein are pharmaceutical compositions comprising a binding protein such as an GFRAL antibody as described herein.

The present disclosure also provides isolated nucleic acid molecules encoding an immunoglobulin heavy chain, an immunoglobulin light chain, VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of binding proteins (e.g., anti-GFRAL antibodies) that bind to a GFRAL polypeptide, a GFRAL polypeptide fragment, or a GFRAL epitope (e.g., one or more amino acids of a GFRAL protein, including of an extracellular domain of a GFRAL protein). In some embodiments, the nucleic acid molecule encodes a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of a monoclonal antibody designated as 1C1, 3P10, 12A3, 5F12, 5A20, 8D8, 17J16, 25M22, 2B8, 22N5, 2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9, 24G2, 6G9, 2B11, 1A3, P1B6, P1H8, or P8G4 as described herein, or a humanized variant thereof. In some embodiments, the nucleic acid molecule further encodes a scaffold region or a framework region, including VH FR1, VH FR2, VH FR3, VH FR4, VL FR1, VL FR2, VL FR3, and/or VL FR4 of a human immunoglobulin amino acid sequence or a variant thereof. Also provided herein are vectors and host cells comprising the nucleic acid molecules encoding a binding protein such as anti-GFRAL antibody, as well as methods of producing a binding protein such as an anti-GFRAL antibody by culturing the host cells provided herein under conditions that promote the production of a binding protein such as an anti-GFRAL antibody.

The present disclosure also provides methods of treating, preventing or alleviating a GFRAL-mediated disease, disorder, or condition, including a GDF15-mediated disease, disorder or condition, (e.g., one or more symptoms) comprising administering to a subject a therapeutically effective amount of a binding protein such as an anti-GFRAL antibody provided herein, including a subject in need thereof, thereby treating, preventing or alleviating the disease, disorder or condition. In some embodiments, the disease, disorder or condition is caused by or otherwise associated with a GDF15 protein (e.g., a human GDF15 protein) and/or a GFRAL protein (e.g., a human GFRAL protein), such as those related to GDF15-induced signaling in a subject. In certain embodiments, the disease, disorder, or condition is treatable by reducing the occurrence, frequency or severity of cachexia, sarcopenia, or muscle wasting, bone wasting or involuntary loss of body weight. In certain embodiments, the disease, disorder, or condition is cachexia. In certain embodiments, the disease, disorder, or condition is a cancer. In certain embodiments, the disease, disorder, or condition is a cardiovascular disease. In certain embodiments, the disease, disorder, or condition is a chronic inflammatory disease (e.g., chronic renal disease, chronic obstructive pulmonary disease). In certain embodiments, the disease, disorder, or condition is a cancer that has decreased sensitivity to (e.g., resistance to) a chemotherapeutic agent (e.g., an anti-tumor antibody such as trastuzumab) that is induced by or related to a GDF15 protein, including elevated levels of GDF15.

In some embodiments, the disease, disorder or condition is or is related to cachexia, sarcopenia, muscle wasting or loss of muscle mass, bone wasting, involuntary loss of body weight (e.g., body weight loss associated with or due to a disease, disorder, or condition). In some embodiments, the disease, disorder or condition is selected from the group of underlying diseases associated with cachexia including, but are not limited to, cancer, chronic renal disease, chronic obstructive pulmonary disease, AIDS, tuberculosis, chronic inflammatory diseases, sepsis and other forms of systemic inflammation, muscle wasting, such as muscular dystrophy, and the eating disorder known as anorexia nervosa.

In some embodiments, the methods of treating, preventing or ameliorating include methods of improving body weight gain or reducing body weight loss, or improving muscle mass gain or reducing muscle mass loss. In some embodiments, the methods of treating, preventing or ameliorating result in improved methods of treating cancer, by preventing, minimizing or reducing the occurrence, frequency or severity of cachexia, sarcopenia or muscle wasting, bone wasting or involuntary loss of body weight.

The present disclosure also provides methods for detecting GFRAL in a sample comprising contacting the sample with a binding protein such as an anti-GFRAL antibody as described herein, that comprises a detectible agent. In certain embodiments, the sample comprises a cell expressing GFRAL on its surface.

The present disclosure also provides kits comprising a binding protein such as an anti-GFRAL antibody that binds to a GFRAL polypeptide, a GFRAL fragment or a GFRAL epitope as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a sequence alignment between various GFRAL proteins. SEQ ID NOS are noted in parenthesis and bold text.

FIG. 2 shows a sequence alignment between various GDF15 proteins. SEQ ID NOS are noted in parenthesis and bold text.

FIG. 3 depicts results of an experiment showing binding affinity of exemplary anti-GFRAL antibodies 1C1 and 3P10 for a GFRAL protein.

FIG. 4A-4B show alignments of VH and VL sequences for exemplary anti-GFRAL antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIG. 5A-5F show alignments of VH and VL sequences for exemplary anti-GFRAL antibodies that bind to domain 1 (FIGS. 5A-5B), domain 2 (FIGS. 5C-5D), or domain 3 (FIGS. 5E-5F) of GFRAL. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 6A-6B show alignments of VH and VL sequences of humanized 1C1 antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 7A-7B show alignments of VH and VL sequences of humanized 25M22 antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 8A-8B show alignments of VH and VL sequences of humanized 17J16 antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 9A-9B show alignments of VH and VL sequences of humanized 5F12 antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIGS. 10A-10B show alignments of VH and VL sequences of humanized 3P10 antibodies. SEQ ID NOS are noted in parenthesis and bold text.

FIG. 11 depicts results of a receptor antagonist assay using a humanized GFRAL antibody.

FIG. 12 depicts results for an Elk1 reporter assay showing of humanized antibodies.

FIGS. 13A-13C depict results of an experiment showing specificity of an exemplary humanized anti-GFRAL antibody.

FIGS. 14A-14B depict results of an experiment showing anti-GFRAL antibody inhibition of GDF15-induced weight loss in DIO mice. For FIG. 14A, from left to right in the figure, the administered treatment was PBS, 3P10, 1C1, 17J16, 5A20, 25M22, 5F12, and 1M03, respectively (for d1, d2 and d3, respectively). For FIG. 14B, from left to right in the figure, the administered treatment was PBS, 8D8 and 12A3, respectively (for d1, d2 and d3, respectively).

FIG. 15 depicts results of an experiment showing anti-GFRAL antibodies in a model of GDF15-induced weight loss.

FIG. 16 depicts results of an experiment showing the effects of anti-GFRAL antibodies on food intake.

FIG. 17 depicts results of an experiment showing the effects of anti-GFRAL antibodies on body weight in DIO mice.

FIG. 18 depicts results of an experiment showing the effects of anti-GFRAL antibodies on food intake in DIO mice.

FIG. 19 depicts results of an experiment showing the effects of anti-GFRAL antibodies on fat mass.

FIG. 20 depicts results of an experiment showing the effect of anti-GFRAL antibodies on GDF15-induced loss of body weight and loss of fat and lean mass in DIO mice.

FIG. 21 depicts results of an experiment showing the effects of anti-GFRAL antibodies on GDF15-induced increase in energy expenditure and GDF15-induced reduction in food intake in DIO mice.

FIG. 22 depicts results of an experiment showing the effects of anti-GFRAL antibodies on GDF15-induced loss of body weight and loss of fat and lean mass in lean mice.

FIG. 23 depicts results of an experiment showing the effects anti-GFRAL antibodies on GDF15-induced change in RER and GDF15-induced reduction in food intake in lean mice.

FIG. 24 depicts results of an experiment showing the effects of an anti-GFRAL antibody (3P10) on body weight in lean mice.

FIG. 25 depicts results of an experiment showing the effects of administration of anti-GFRAL antibodies on food intake in lean mice.

FIGS. 26A-26C depict results of an experiment showing the effects of dietary adenine.

FIG. 27A depicts results of an experiment showing the effects of dietary adenine.

FIG. 27B depicts results of an experiment showing the effects of an exemplary an anti-GFRAL antibody (3P10) on mice with chronic kidney damage.

FIG. 28 depicts results of an experiment showing the effects of an exemplary an anti-GFRAL antibody (3P10) on mice with chronic kidney damage.

FIG. 29 depicts results of an experiment showing the effects of an exemplary humanized anti-GFRAL antibody (h3P10) of GDF15-induced body weight loss.

FIG. 30 shows an exemplary crystal of a complex of a GFRAL protein and a GDF15 protein.

FIG. 31 illustrates an exemplary GFRAL electron density map.

FIG. 32 shows an exemplary ribbon diagram of a GFRAL/GDF15 complex formed in an asymmetric GFRAL/GDF15 crystal unit. GFRAL protein domains D2 and D3 are indicated as GFRAL D2 and GFRAL D3.

FIG. 33 shows an exemplary ribbon diagram of a dimer of two GFRAL/GDF15 complexes. The GFRAL protein domains D2 and D3 are indicated as GFRAL D2 and GFRAL D3.

FIGS. 34A-34B show different surface representations of a dimer of two GFRAL/GDF15 complexes.

FIG. 35 illustrates GFRAL amino acid residues interacting with GDF15 amino residues.

FIGS. 36A-36D illustrate a GFRAL/GDF15 interface. The GFRAL protein domains D2 and D3 are indicated as GFRAL D2 and GFRAL D3.

FIGS. 37A-37B show different aspects of a superposition of a GFRAL protein and GFRα1 depicted as ribbon diagrams.

FIGS. 38A-38D illustrate different aspects of the interaction of a GFRAL protein with a RET protein in a RET/GFRAL/GDF15 model.

FIGS. 39A-39B illustrate amino acid residues on the RET protein interface of a GFRAL protein.

FIG. 40 illustrates exemplary crystals of a complex having a GFRAL protein, a 3P10 Fab and a 25M22 Fab produced under crystallization conditions B11, D11, and H8.

FIG. 41 illustrates exemplary crystals of a complex having a GFRAL protein, a 8D8 Fab and a 5F12 Fab produced under crystallization conditions C6, E11, and C2.

FIG. 42 illustrates exemplary electron density maps of 3P10 Fab and 25M22 Fab CDR regions in the crystal structure of a GFRAL/3P10/25M22 Fab complex.

FIG. 43 illustrates an exemplary electron density of a GFRAL/8D8/5F12 Fab complex.

FIG. 44 shows an exemplary ribbon diagram of a GFRAL/3P10/25M22 Fab complex formed in an asymmetric GFRAL/3P10/25M22 Fab complex crystal unit.

FIG. 45 shows alignments of 3P10 Fab and 25M22 Fab CDR sequences (top lines) with GFRAL amino acid residues (bottom lines) that are involved in 3P10 Fab and 25M22 Fab binding. Residues involved in the GFRAL-Fab interaction are boxed. For the 3P10 Fab, amino acid residues Q1 to S120 of SEQ ID NO: 1824 are shown for the Hc and amino acid residues D1 to F120 of SEQ ID NO: 1825 are shown for the Lc. For the 25M22 Fab, amino acid residues Q1 to S120 of SEQ ID NO: 1826 are shown for the Hc.

FIG. 46 shows a ribbon diagram illustrating the interaction of a GFRAL 3P10 Fab epitope and a 3P10 Fab heavy chain CDR region.

FIG. 47 shows a ribbon diagram illustrating the interaction of a GFRAL 3P10 Fab epitope and a 3P10 light chain CDR region.

FIG. 48 shows a ribbon diagram illustrating the interaction of a GFRAL 25M22 epitope and a 25M22 Fab heavy chain CDR region.

FIG. 49 shows amino acid sequences for a GFRAL protein (residues S130 to N318 of SEQ ID NO: 1797) and 25M22 Fab heavy chain (HC) residues Q1 to P138 and G146 to P225 of SEQ ID NO:1826 and light chain (LC) residues D1 to E218 of SEQ ID NO:1827. Residues having grey background and white text indicate core interaction interface amino acids for both the GFRAL protein and the 25M22 Fab. Residues on the Fab HC or LC having grey background with black or white lettering indicate exemplary CDR sequences for the 25M22 Fab.

FIGS. 50A-50B illustrate core interaction interface amino acid residues on a GFRAL protein and on 25M22 Fab involved in the GFRAL/25M22 Fab interaction.

FIG. 51 illustrates boundary interaction interface amino acid residues on a GFRAL protein involved in the GFRAL/25M22 Fab interaction.

FIG. 52 shows exemplary side views of a ribbon diagram illustrating overlapping 25M22 Fab and GDF15 epitopes on a GFRAL protein as space-filled surface models (core interaction interface amino acids).

FIG. 53 shows a top view of a ribbon diagram illustrating overlapping 25M22 Fab and GDF15 epitopes on a GFRAL protein as space-filled surface models (core interaction interface amino acids).

FIG. 54 shows amino acid sequences for a GFRAL protein (residues S130 to N318 of SEQ ID NO: 1797) and 3P10 Fab heavy chain (HC) residues Q1 to A130 and G138 to C221 of SEQ ID NO:1824 and light chain (LC) residues D1 to C218 of SEQ ID NO:1825. Residues having grey background and white text indicate core interaction interface amino acids for both the GFRAL protein and the 3P10 Fab. Residues on the Fab HC or LC having grey background with black or white lettering indicate exemplary CDR sequences for the 3P10 Fab.

FIGS. 55A-55B show a ribbon diagram illustrating a crystal structure of a GFRAL/3P10 Fab complex. Interaction interface residues are shown as stick models (FIG. 55A) or space-filled surface models (FIG. 55B).

FIGS. 56A-56B illustrates interface residues of a GFRAL 3P10 Fab epitope as space-filled surface models in a ribbon diagram of GFRAL. FIG. 56A shows core interaction interface residues and FIG. 56B shows boundary interaction interface residues.

FIG. 57 illustrates overlapping residues of a GFRAL protein that bind to 3P10 Fab and residues of a GFRALprotein that bind to a RET protein as space-filled surface models on a ribbon diagram of a GFRALprotein.

FIG. 58 illustrates the combined coverage of boundary interaction interface residues of a GFRAL protein that bind to 3P10 Fab and 25M22 Fab.

FIG. 59 shows an exemplary ribbon diagram of a GFRAL/8D8/5F12 Fab complex formed in an asymmetric GFRAL/8D8/5F12 Fab complex crystal unit.

FIG. 60 shows an exemplary ribbon diagram of the 8D8 Fab and 5F12 Fab binding sites on a GFRAL protein. Residues on the GFRAL protein that are important for Fab binding are shown as stick models.

FIG. 61 shows amino acid sequences for a GFRAL protein (residues S130 to N318 of SEQ ID NO: 1797) and 8D8 Fab heavy chain (HC) residues Q1 to K217 of SEQ ID NO:1828 and light chain (LC) residues D1 to R211 of SEQ ID NO:1829. Residues having grey background and white text indicate core interaction interface amino acids for both the GFRAL protein and the 8D8 Fab. Residues on the Fab HC or LC having grey background with black or white lettering indicate exemplary CDR sequences for the 8D8 Fab.

FIGS. 62A, 62B, 62C and 62D illustrate core and boundary amino acid residues in a GFRAL/8D8 Fab interaction interface.

FIGS. 63A, 63B, 63C and 63D illustrate the core and boundary amino acid residues in a GFRAL/5F12 Fab interaction interface.

FIG. 64 shows amino acid sequences for a GFRAL (residues S130 to N318 of SEQ ID NO: 1797) and 5F12 Fab heavy chain (HC) residues Q1 to K223 of SEQ ID NO:1830 and light chain (LC) residues N1 to E217 of SEQ ID NO:1831. Residues having grey background and white text indicate core interaction interface amino acids for both the GFRAL protein and the 5F12 Fab. Residues on the Fab HC or LC having grey background with black or white lettering indicate exemplary CDR sequences for the 5F12 Fab.

DETAILED DESCRIPTION

Binding proteins, such as antibodies that bind a GFRAL protein, including a human GFRAL protein, are provided herein. A unique property of such binding proteins, including antibodies disclosed herein, is their antagonistic nature, including the ability to inhibit an effect of a GDF15 protein and/or to inhibit binding of a GDF15 protein to a GFRAL protein or inhibit binding of a RET protein to a GFRAL protein, including wherein the inhibition of binding reduces (e.g., blocks) GDF15 signaling. Remarkably and specifically, binding proteins such as antibodies to a GFRAL protein disclosed herein (i) bind to a GFRAL protein, (ii) inhibit binding of a GDF15 protein to a GFRAL protein, and/or (iii) inhibit binding of a RET protein to a GFRAL protein, including blocking the formation of a GDF15/GFRAL protein complex or a GDF15/GFRAL/RET protein complex or GDF15 signaling, including, for example, as measured by several in vitro cell-based assays. Such assays may include (1) a ELK1-luciferase reporter assay (see, e.g., Example 3); and/or (2) ERK-phosphorylation assay in U2OS cells (see, e.g., Example 4). Binding proteins such as anti-GFRAL antibodies, as described herein, therefore are expected to inhibit GDF15 activities in vivo (e.g., related to the signaling function of GDF15). This property makes the disclosed binding proteins, including anti-GFRAL antibodies, viable therapeutics for the treatment of a disease, disorder or condition that is caused by or otherwise associated with a GDF15 protein (e.g., a human GDF15 protein) and/or a GFRAL protein (e.g., a human GFRAL protein), such as those related to GDF15-induced signaling in a subject.

The binding proteins, such as antibodies that bind a GFRAL protein, that are provided herein share the common feature of antagonizing the binding of (i) a GDF15 protein to a GFRAL protein and/or (ii) a RET protein to a GFRAL protein. The anti-GFRAL antibodies provided herein include humanized anti-GFRAL antibodies, including humanized anti-GFRAL antibodies derived from or based on 1C1, 3P10, 12A3, 5F12, 5A20, 8D8, 17J16, 25M22, 2B8, 22N5, 2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9, 24G2, 6G9, 2B11, 1A3, P1B6, P1H8, and/or P8G4 having CDR sequences as described in Tables 1-24 or FIGS. 4-10. Such anti-GFRAL antibodies, including humanized anti-GFRAL antibodies, bind to a specific domain of a GFRAL protein including, for example, those that bind to: (i) domain 1 of a GFRAL protein (e.g., amino acid residues Q20 to S130 of SEQ ID NO: 1797); (ii) domain 2 of a GFRAL protein (e.g., amino acid residues C131 to C210 of SEQ ID NO: 1797); (iii) domain 3 of a GFRAL protein (e.g., amino acid residues C220 to C316 of SEQ ID NO: 1797); or (iv) an extracellular domain of a GFRAL protein (e.g., amino acid residues Q20 to E351 of SEQ ID NO: 1797).

In some embodiments of the present disclosure, the binding proteins such as anti-GFRAL antibodies may comprise immunoglobulin variable regions which comprise one or more complementary determining regions (CDRs) as described in Tables 1-24. In such binding proteins (e.g., anti-GFRAL antibodies), the CDRs may be joined with one or more scaffold regions or framework regions, which orient(s) the CDR(s) such that the proper antigen binding properties of the CDR(s) is achieved. Such binding proteins, including anti-GFRAL antibodies as described herein, can inhibit (e.g., block) the interaction (i) between a GDF15 protein and a GFRAL protein and/or (ii) between a RET protein and a GFRAL protein. Such binding proteins, including anti-GFRAL antibodies as described herein, can inhibit (e.g., block) GDF15 signaling.

In some embodiments of the present disclosure, the binding proteins such as anti-GFRAL antibodies bind to: (i) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues SER156, GLN147, LEU148, ALA149, SER150, TYR151, LEU152, LYS153, ALA154, CYS155, PHE174, TYR175, GLU136, ALA137, CYS138, VAL139, GLY140, ASP141, VAL142, VAL143, CYS144, ASN145, ALA146, LEU186, CYS189, CYS191, ALA192, GLN193, SER194, ASP195, ILE196, PRO197, CYS198, GLN199, GLN200, SER201, LYS202, GLU203, ALA204, LEU205, HIS206, SER207, SER130, CYS131, LEU132, GLU133, VAL134, orALA135 of a GFRAL protein (SEQ ID NO: 1797); (ii) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues LEU132, GLU133, VAL134, ALA135, GLU136, ALA137, CYS138, VAL139, GLY140, ASP141, VAL142, VAL143, CYS144, ASN145, ALA146, GLN147, LEU148, ALA149, SER150, TYR151, PHE174, TYR175, ALA169, ALA170, ILE171, ARG172, PHE173, GLN176, ASN177, ILE178, PRO179, PHE180, ASN181, ILE182, ALA183, GLN184, MET185, LEU186, ALA187, PHE188, or CYS189 of SEQ ID NO: 1797; (iii) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues LEU164, LYS208, VAL212, ASN213, MET214, VAL215, PRO216, PRO217, PRO218, THR219, CYS220, LEU221, VAL223, TRP245, LEU267, CYS269, GLN28, VAL289, GLN290, CYS291, THR292, CYS293, ARG294, THR295, ILE296, THR297, GLN298, SER299, GLU300, GLU301, SER302, LEU303, CYS304, LYS305, ILE306, PHE307, GLN308, HIS309, MET310, LEU311, HIS312, ARG313, LYS314, SER315, CYS316, or PHE317 of SEQ ID NO: 1797; (iv) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues CYS233, ARG234, ARG235, HIS236, TYR237, ARG238, THR239, PHE240, GLN241, SER242, LYS243, CYS244, TRP245, GLN246, ARG247, VAL248, THR249, ARG250, LYS251, CYS252, HIS253, GLU254, ASP255, GLU256, ASN257, CYS258, ILE259, SER260, THR261, LEU262, SER263, LYS264, ASP266, LEU267, THR268, SER272, ASP274, CYS275, ALA278, CYS269, SER270, SER302, LEU303, ILE306, HIS309, LEU311, MET310, SER315, or CYS316 of SEQ ID NO: 1797; (v) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues GLY140, LEU148, ALA149, ALA146, VAL142, ASN145, VAL139, ALA135, GLU136, LEU152, LEU132, SER201, ALA204, LEU205, LYS153, ILE196, PRO197, or GLN200 of SEQ ID NO: 1797; (vi) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues GLU136, ALA137, VAL139, GLY140, ASP141, VAL142, VAL143, CYS144, ASN145, ALA146, GLN147, PHE173, ASN177, ILE178, PRO179, ASN181, ILE182, or MET185 of SEQ ID NO: 1797; (vii) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues GLN298 or GLU301 of SEQ ID NO: 1797; or (viii) an epitope of a GFRAL protein comprising at least one of (e.g., one or more) amino acid residues ARG234, ARG238, GLN241, SER242, LYS243, TRP245, GLN246, THR249, ARG250, LYS251, CYS252, HIS253, ASP255, ASN257, CYS258, SER260, THR261, or LEU262 of SEQ ID NO: 1797. Such antibodies provided above can, in some embodiments, inhibit GDF15-induced signaling and/or signaling or activation of a GFRAL/GDF15 or RET/GFRAL/GDF15 receptor complex, for example, in a cell that expresses a GFRAL protein. Additionally, in some embodiments of the present disclosure, the antibody is a monoclonal antibody, for example, a humanized antibody.

General Techniques

Techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds., (2003)); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Z. An, ed, Wiley, Hoboken N.J. (2009); Monoclonal Antibodies: Methods and Protocols, M. Albitar, ed., Humana Press, Totawa, N.J. (2010); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Kontermann and Dubel, eds., Springer-Verlag, Heidelberg, 2010.

Terminology

Unless described otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.

The term “GDNF Family Receptor Alpha Like” “growth differentiation factor 15 receptor,” “GFRAL” or “GFRAL protein” and similar terms refers to a polypeptide (“polypeptide,” and “protein” are used interchangeably herein) or any native GFRAL from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkey (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated, and, in certain embodiments, includes related GFRAL polypeptides, including SNP variants thereof. GFRAL is also known in the art as “C6orf144,” “Chromosome 6 Open reading Frame 144,” “BA360D14.1” “IVF19356,” and “UNQ9356.”

The amino acid sequence of a full-length precursor human GFRAL is provided below, which includes a signal peptide sequence (underlined and lowercase residues):

(SEQ ID NO: 1797) mivfiflamglsleneytsQTNNCTYLREQCLRDANGCKHAWRVMEDAC NDSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLG KKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQ LASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDI PCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCW QRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCR TITQSEESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTG FHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSSIQIPG EL.

The amino acid sequence of a mature human GFRAL polypeptide is provided below:

(SEQ ID NO: 1798) QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSY CNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFK WNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNP CDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCA VNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCI STLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQH MLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMC MTVTCGILLLVMVKLRTSRISSKARDPSSIQIPGEL.

In some embodiments, GFRAL refers to a protein that is at least 55% identical to the amino acid sequence of mature human GFRAL (SEQ ID NO: 1798). Binding proteins, such as anti-GFRAL antibodies as disclosed herein, can bind GFRAL and/or modulate signaling, as described herein. In certain embodiments, antibodies described herein bind to human GFRAL.

Human GFRAL has an extracellular domain (e.g., residues 20-351 of SEQ ID NO: 1797), a transmembrane domain (e.g., residues 352-371 of SEQ ID NO: 1797) and a cytoplasmic domain (e.g., residues 372-394 of SEQ ID NO: 1797).

A nucleic acid sequence encoding a precursor GFRAL polypeptide is provided below:

(SEQ ID NO: 1799) TTATTCTGGACAGTTACTCTTAAGAAAGTTGTCAGAAGAAACGCATCT GCCTTTTTTTCCAGGTGAACTGCCGTGAGTTGTCCAGCATGATAGTG TTTATTTTCTTGGCTATGGGGTTAAGCTTGGAAAATGAATACACTTCC CAAACCAATAATTGCACATATTTAAGAGAGCAATGCTTACGTGATGCA AATGGATGTAAACATGCTTGGAGAGTAATGGAAGATGCCTGCAATGA TTCAGATCCAGGTGACCCCTGCAAGATGAGGAATTCATCATACTGTA ACCTGAGTATCCAGTACTTAGTGGAAAGCAATTTCCAATTTAAAGAGT GTCTTTGCACTGATGACTTCTATTGTACTGTGAACAAACTGCTTGGAA AAAAATGTATCAATAAATCAGATAACGTGAAAGAGGATAAATTCAAAT GGAATCTAACTACACGTTCCCATCATGGATTCAAAGGGATGTGGTCC TGTTTGGAAGTGGCAGAGGCATGTGTAGGGGATGTGGTCTGTAATGC ACAGTTGGCCTCTTACCTTAAAGCTTGCTCAGCAAATGGAAATCCGT GTGATCTGAAACAGTGCCAAGCAGCCATACGGTTCTTCTATCAAAATA TACCTTTTAACATTGCCCAGATGTTGGCTTTTTGTGACTGTGCTCAAT CTGATATACCTTGTCAGCAGTCCAAAGAAGCTCTTCACAGCAAGACA TGTGCAGTGAACATGGTTCCACCCCCTACTTGCCTCAGTGTAATTCG CAGCTGCCAAAATGATGAATTATGCAGGAGGCACTATAGAACATTTC AGTCAAAATGCTGGCAGCGTGTGACTAGAAAGTGCCATGAAGATGAG AATTGCATTAGCACCTTAAGCAAACAGGACCTCACTTGTTCAGGAAGT GATGACTGCAAAGCTGCTTACATAGATATCCTTGGGACGGTCCTTCA AGTGCAATGTACCTGTAGGACCATTACACAAAGTGAGGAATCTTTGT GTAAGATTTTCCAGCACATGCTTCATAGAAAATCATGTTTCAATTATCC AACCCTGTCTAATGTCAAAGGCATGGCATTGTATACAAGAAAACATGC AAACAAAATCACTTTAACTGGATTTCATTCCCCCTTCAATGGAGAAGT AATCTATGCTGCCATGTGCATGACAGTCACCTGTGGAATCCTTCTGTT GGTTATGGTCAAGCTTAGAACTTCCAGAATATCAAGTAAAGCAAGAG ATCCTTCATCGATCCAAATACCTGGAGAACTCTGATTCATTAGGAGTC ATGGACCTATAACAATCACTCTTTTCTCTGCTTTTCTTCTTTCCTCTTT TCTTCTCTCCTCTCCTCTCCTCTCTTCTCCTCTCCTCCCCTCCCCTCT CTGTTTCTTTTTCTTTTTCTTTTCTTTTTTGTGGCGGAGTTTTGCTCTTG TTGCCCAGGCTGCAGTACAATGGCTCAATCTCGGTTCACTGCAACCT CTGCCTCCAAGGTTCAAGTGATTTTCCTGCCTCAGCCTTCCCGAGTA GCTGGGATTACAGGTACCCGCCACCACGCCCAGCTAATTTTTTTGTA TTTTTAGTAGAGATGGGGTTTTGCCAAATTGGCCAGGGTGGTCTCAA ACTCCTGACCTCAGGTGATCCACCCACCTCGGCCTCCCAAAGTGCTG GGATTACAGGCGTGAGCAACCACGTCAAGACAACAATCACTTTCTTT AAAGCAAATCCTACAGCTGGTCAACACCCTATTCCATCTGTCATCGA GAAAGAAAATGTTAAAATAGACTTAAAAATATTGCTTTGTTACATATAA TAATATGGCATGATGATGTTATTTTTTTCTTAATACTCAAGAAAAAATA TATGGTGGTATCTTTTACAACACTGGAACAGAAATAAAGTTTCCCTTG AAGGC.

“GFRAL” as used herein encompasses human GFRAL and variants thereof, including but not limited to orthologs thereof, such as murine GFRAL, rat GFRAL, cyno GFRAL, and the like. GFRAL is not TGFβ RII (NCBI Ref. Seqs.: NM_001024847.2 (GI:133908632); NM_003242.5 (GI:133908633)) or orthologs thereof. GFRAL is distinct from TGFβ RI (NCBI Ref. Seqs.: NP_001124388.1 (GI:195963412); NP_004603.1 (GI:4759226)) or orthologs thereof. In certain embodiments, GFRAL may be a protein having the amino acid sequence that is at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1797. Such exemplary GFRAL proteins include chimpanzee (99%), cynomolgus monkey (92%), giant panda (82%), dog (81%), cat (80%), pig (77%), bovine (75%), mouse (70%), rat (70%), Chinese hamster (65%), and platypus (59%), as shown in FIG. 1.

An amino acid sequence of a GFRAL protein from cynomolgus monkey (cyno), scientific name Macaca fascicularis, is provided below, which includes a signal peptide sequence (underlined residues):

(SEQ ID NO: 1800) mivliflalglsleneytsQTNNCTYLREQCLHDANGCKHAWRIMEDAC NDSDPGDPCKMNNSSYCNLSIQYLVESNFRFKECLCTDDFYCTVNKLLG KECVNKSDNMREDKFKWNLTTHSHHGFKGMWSCLEVAEACVGDVVCNAQ LASYLKACSANGNPCDVKHCQAAIRFFYQNIPFNIAQMLAFCDCSQSDI PCQQSKEALHSKPCALNMVPPPTCLNVIRSCQNDELCRRHYRTFQSKCW QRVTRKCHEDENCISALSKQDLTCSGSDDCKAAYIDILGTVLQVQCNCR TITQSEESLCKIFQHMLHRKSCFNYPTLSNVKSMALYTRKHTNKITLTG FQSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSLSQVPG EL.

An encoding nucleic acid sequence of a cyno GFRAL protein is provided below:

(SEQ ID NO: 1801) ACCCACCAGAAAGAAGGAGCTCCAGACACATCTGAACGTCTGAAGG AAGAAACTCCCGACACACCATCTTTAAGAAATGTAACTCTCACTGCGA GGGTATGTGGCTTCATTCTTGAAGTCAGGGAGACCAAGAACCCACCA ATTGCAGGCACACAAGGGGTCCTTATTTTATTCAGGTGAACAGCTGT GAGTTGTCCAGCATGATAGTGCTTATTTTCTTGGCTTTGGGGCTAAGC TTGGAAAATGAATACACTTCCCAAACCAATAATTGCACATATTTAAGA GAGCAATGCCTACATGATGCAAATGGATGTAAACATGCTTGGAGAAT AATGGAAGATGCCTGCAATGATTCAGATCCAGGTGACCCCTGCAAGA TGAATAATTCATCATACTGTAACCTGAGTATCCAGTACTTAGTGGAAA GCAATTTCCGATTTAAAGAGTGTCTTTGCACTGATGACTTCTATTGTA CTGTGAACAAACTGCTTGGAAAAGAATGTGTCAATAAATCAGATAACA TGAGAGAGGATAAATTCAAATGGAATCTAACTACACATTCCCATCATG GATTCAAAGGGATGTGGTCCTGTTTGGAAGTGGCAGAGGCATGTGTA GGGGATGTGGTCTGTAATGCACAGTTGGCCTCTTACCTTAAAGCTTG CTCAGCAAATGGAAATCCGTGTGATGTGAAACACTGCCAAGCAGCCA TACGGTTCTTCTATCAAAATATACCTTTTAACATTGCCCAGATGTTGG CTTTTTGTGACTGTTCTCAATCTGATATACCTTGTCAGCAGTCCAAAG AAGCTCTTCACAGCAAGCCATGTGCACTGAACATGGTTCCACCCCCT ACTTGCCTCAATGTAATTCGCAGCTGCCAAAATGATGAATTATGCAGG AGGCACTATAGAACATTTCAGTCAAAATGCTGGCAGCGTGTGACTAG AAAGTGCCATGAAGATGAGAATTGCATTAGCGCCTTAAGCAAACAGG ACCTCACATGTTCAGGAAGTGATGACTGCAAAGCTGCTTACATAGAT ATCCTTGGGACAGTCCTTCAAGTGCAATGTAACTGTAGGACCATTAC ACAAAGTGAGGAATCTTTGTGCAAGATTTTCCAGCACATGCTTCATAG AAAATCATGTTTCAATTATCCAACCCTGTCTAATGTCAAAAGCATGGC ATTGTATACAAGAAAACATACAAACAAAATCACTTTAACTGGATTTCAG TCCCCCTTCAATGGAGAAGTAATCTATGCTGCCATGTGCATGACAGT CACCTGTGGAATCCTTCTCTTGGTTATGGTCAAGCTTAGAACTTCCAG AATATCAAGTAAAGCAAGAGATCCTTCACTGAGCCAAGTACCTGGAG AACTCTGATTCATTAGGAGTCATGGACCCATAACAATCACTCCCCTTC CCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCTTCCCT TCC.

An amino acid sequence of a GFRAL protein from mouse, scientific name Mus musculus, is provided below, which includes a signal peptide sequence (underlined residues):

(SEQ ID NO: 1802) mlvfiflavtlssenesssQTNDCAHLIQKCLIDANGCEQSWRSMEDTC LTPGDSCKINNSLHCNLSIQALVEKNFQFKECLCMDDLHCTVNKLFGKK CTNKTDNMEKDNKDKWNLTTTPFYHGFKQMQSCLEVTEACVGDVVCNAQ LALYLKACSANGNLCDVKHCQAAIRFFYQNMPFNTAQMLAFCDCAQSDI PCQQSKETLHSKPCALNIVPPPTCLSVIHTCRNDELCRTHYRTFQTECW PHITGKCHEDETCISMLGKQDLTCSGSESCRAAFLGTFGTVLQVPCACR GVTQAEEHVCMIFQHMLHSKSCFNYPTPNVKDISSYEKKNSKEITLTGF NSFFNGELLYVVVCMAVTCGILFLVMLKLRIQSEKRDPSSIEIAGGVII Q.

An encoding nucleic acid sequence of a mouse GFRAL protein is provided below:

(SEQ ID NO: 1803) AACAATTGAATTTGAATACAATTAGGAAAGTTCACAGCTCAAAACAAA CTGGTGAGGAACAGCTGACACCAGAAGCTGACTCTAATTGGCTGGCT CTTAGGAAGCAAAACCTTTACACAGAAACTTCAGTTGGGATGTTGGTT GGTGTCAGTTCATCCGCCTTTCTCCCAGGGAGACCATCTTGAGTTGT CCAACATGCTAGTGTTCATTTTCCTGGCTGTTACGTTAAGCTCAGAAA ATGAATCCTCTTCCCAAACAAATGATTGTGCACATTTAATACAGAAAT GCTTGATTGATGCAAATGGCTGTGAGCAGTCATGGAGATCAATGGAA GACACCTGCCTTACTCCAGGTGACTCCTGCAAGATAAATAATTCACTA CATTGTAACCTGAGTATCCAGGCTTTGGTGGAAAAAAATTTCCAATTT AAAGAGTGTCTTTGTATGGATGACCTCCACTGTACAGTAAACAAACTT TTTGGAAAAAAGTGCACCAATAAGACAGATAACATGGAAAAGGACAA TAAAGATAAATGGAATCTAACTACTACTCCTTTCTATCATGGATTCAAA CAGATGCAGTCTTGTTTGGAGGTGACAGAGGCGTGTGTAGGGGATG TGGTTTGTAATGCACAGTTGGCCCTTTACCTTAAAGCATGCTCAGCAA ATGGAAATCTGTGTGATGTGAAACACTGCCAAGCAGCCATACGGTTC TTCTATCAAAATATGCCTTTTAACACTGCCCAGATGTTGGCTTTTTGTG ACTGTGCTCAATCTGATATACCCTGTCAGCAATCCAAAGAAACTCTTC ACAGCAAGCCATGTGCACTGAATATAGTTCCACCCCCCACTTGCCTC AGTGTAATTCACACTTGCCGAAATGATGAATTATGCAGGACACACTAC CGAACATTCCAGACAGAATGCTGGCCCCACATAACTGGGAAGTGCCA TGAAGATGAGACCTGCATTAGCATGTTAGGCAAGCAAGACCTTACTT GTTCTGGGAGTGAGAGCTGCAGGGCTGCCTTCCTAGGAACCTTTGG GACAGTCCTGCAAGTACCCTGTGCTTGCAGGGGCGTTACACAGGCT GAAGAACACGTGTGCATGATTTTCCAGCACATGCTTCATAGCAAATC GTGTTTCAATTACCCAACTCCTAATGTCAAAGACATTTCCTCATATGA AAAAAAGAATTCAAAAGAAATTACTCTGACTGGATTCAATTCTTTCTTC AATGGAGAACTACTCTATGTTGTTGTGTGCATGGCAGTTACCTGTGG AATTCTTTTCTTGGTGATGCTCAAGTTAAGGATACAAAGTGAAAAAAG AGATCCCTCATCCATCGAAATAGCTGGAGGTGTCATCATTCAGTGAG CTGCAGATCACTTACCAACCACATGTCTGTGTGACTAACCAATGGAA AATTACATTTGCCAATAACGCAATTTAAGATGGATTTGACAATATTTAG TCATTATATGTAACAGTGACTGGTACAGTAATATACCACAATGATCAC AGATCTGTTTTTGTTTTTGTTTTTAATGTTTGAGTAAATACTTGTTGTG GTGTCATAACTAGTTGATAACATTTTCTTTAAAGACAACAGGTGTCAT GTAAAATGTGACAAATTTGCTGGAAGACTATCAATCCACATATCAACT TCTATCTTATGGAACTAATCATAATTAGTGTGTGCAGTTTTCTGAACAA GGTTATAGTTTTCCATTAAGTTGGTAAAATTAAAATGCTAAGTAGAATA TTGAGTATACTTGTTATTTATATATTCTTACTTAGTGTCCAATCATTAAA CAAATTGGTAACATTGAACATATTTAGTTAGATGACTGCTTATGAAAAT AAGAACTGACATCTTACAAATTTTATAATTTAAATAGTATTGAATTTTAC TTTTTATTTGGTATGTTAAGATTCATAATATATAAAGCAGCTACATTGG TTGAGAAAAGTCAATGGTTACTCCAGTAATGATATACTTTGTGAATTTA TTTATTTTTGCTAATTAATGATCCTGAATGTAATCATGATGAAATAAAA AAGACATACTTAAATTGCT.

An amino acid sequence of a GFRAL protein from rat, scientific name Rattus norvegicus, is provided below, which includes a signal peptide sequence (underlined residues):

(SEQ ID NO: 1804) mlvfiflavrlssenesssQTNDCAYFMRQCLTDTDGCKQSWRSMEDA CLVSGDSCKINNPLPCNLSIQSLVEKHFQFKGCLCTDDLHCTVNKIFG KKCTNKTDSMKKDNKYKRNLTTPLYHDTGFKQMQSCLEVTEACVGDVV CNAQLALYLKACTANGNLCDVKHCQAAIRFFYQNMPFNTAQMLAFCDC AQSDIPCQQSKETLHSKPCALNVVPPPTCLSVIHTCRNDELCRTYYRT FQTECWPHVAGKCREDETCISMLGKQDLTCSGSDSCRAAYLGTFGTVL QVPCACRSITQGEEPLCMAFQHMLHSKSCFNYPTPNVKDISSYERKHS KEITLTGFNSPFSGELIYVVVCMVVTSGILSLVMLKLRIPSKKRDPAP IEIAGAVIIQ.

An encoding nucleic acid sequence of a rat GFRAL protein is provided below:

(SEQ ID NO: 1805) ACAAATGATTGTGCATATTTCATGCGGCAATGCTTGACTGATACAGAT GGCTGTAAGCAGTCATGGAGATCAATGGAAGACGCCTGCCTTGTCTC AGGTGACTCCTGCAAGATAAATAATCCATTGCCTTGTAACCTGAGTAT CCAGTCTTTGGTGGAAAAACATTTTCAATTTAAAGGGTGTCTTTGCAC TGATGATCTCCACTGTACAGTAAACAAAATTTTTGGAAAAAAGTGCAC CAATAAGACAGATAGCATGAAAAAAGATAATAAATACAAACGGAATCT AACTACTCCTTTATATCATGATACAGGATTCAAACAGATGCAGTCTTG TTTGGAAGTGACAGAGGCGTGTGTAGGGGATGTGGTTTGTAATGCAC AGTTGGCCCTTTACCTTAAAGCATGCACAGCAAATGGAAATCTGTGT GATGTGAAACACTGCCAAGCGGCCATACGGTTCTTCTATCAAAATAT GCCTTTTAACACTGCCCAGATGTTGGCTTTTTGTGACTGTGCTCAATC TGATATACCCTGTCAACAATCCAAAGAAACTCTTCACAGCAAGCCATG TGCACTGAACGTAGTTCCACCCCCCACTTGCCTCAGTGTAATTCACA CTTGCCGAAATGATGAATTATGCAGGACATACTACCGAACATTCCAG ACAGAATGCTGGCCCCATGTGGCTGGGAAGTGTCGTGAAGATGAGA CCTGCATTAGTATGCTGGGCAAGCAAGACCTTACTTGTTCTGGGAGT GACAGCTGCAGGGCAGCCTACCTAGGAACCTTCGGGACAGTCCTTC AGGTGCCGTGTGCTTGCAGAAGCATCACACAGGGTGAAGAACCCTT GTGCATGGCTTTCCAGCACATGCTTCACAGCAAATCATGTTTCAATTA CCCAACTCCTAATGTCAAAGACATTTCCTCATATGAAAGAAAGCATTC AAAAGAAATTACCCTGACTGGATTCAATTCTCCCTTCAGTGGAGAACT AATCTATGTTGTTGTGTGCATGGTAGTTACCAGCGGGATTCTTTCCTT GGTGATGCTCAAGCTAAGGATACCTAGTAAGAAAAGAGACCCCGCG CCCATCGAAATAGCTGGAGCTGTCATCATTCAGTGA.

A GFRAL protein or GFRAL also refers to a protein that has one or more alteration in the amino acid residues (e.g., at locations that are not conserved across variants and/or species) while retaining the conserved domains and having a biological activity similar to the naturally-occurring GFRAL. GFRAL may be encoded by nucleic acid sequences that vary in one or more bases from a naturally-occurring DNA sequence but still translate into an amino acid sequence that corresponds to the a naturally-occurring protein due to degeneracy of the genetic code. A GFRAL protein also refers to a protein that differs from the naturally-occurring sequences of GFRAL by one or more conservative substitutions and/or tags and/or conjugates.

The term “GFRAL” or “GFRAL protein” encompasses “full-length” unprocessed GFRAL as well as any form of GFRAL that results from processing in the cell. The term GFRAL or “GFRAL protein” also includes: allelic variants (e.g., SNP variants); splice variants; isoforms; fragments; derivatives; substitution, deletion, and insertion variants; fusion polypeptides; and interspecies homologs, preferably, which retain GFRAL activity and/or are sufficient to generate an anti-GFRAL immune response. As those skilled in the art will appreciate, an anti-GFRAL antibody provided herein can bind to a GFRAL protein, including a GFRAL polypeptide fragment, a GFRAL antigen, and/or a GFRAL epitope. An epitope may be part of a larger GFRAL antigen, which may be part of a larger GFRAL polypeptide fragment, which, in turn, may be part of a larger GFRAL protein. A GFRAL protein may exist in a native or denatured form. GFRAL proteins described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. A GFRAL protein may comprise a polypeptide having the same amino acid sequence as a corresponding GFRAL polypeptide derived from nature. GFRAL proteins encompass truncated or secreted forms of a GFRAL polypeptide (e.g., an extracellular domain sequence), variant forms (e.g., alternatively spliced forms) and allelic variants of the polypeptide. GFRAL polypeptides described herein (e.g., human GFRAL) may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.

A GFRAL protein can lack at least 5, at least 10, up to at least 50 or more amino acids relative to a naturally-occurring full-length GFRAL polypeptide. For example, the GFRAL protein may not contain the signal sequence based on the amino acid sequence of a naturally-occurring GFRAL polypeptide. A GFRAL protein may also contain the same or similar post-translational modifications as a naturally-occurring GFRAL polypeptide or may not contain a post-translational modification. For example, the protein may have the same or similar glycosylation pattern as those of a naturally-occurring GFRAL polypeptide or may contain no glycosylation. In other embodiments, the GFRL protein includes mutations relative to the sequence of naturally-occurring GFRAL protein that introduce a glycosylation site at a location not present in the naturally-occurring GFRAL protein.

In certain embodiments, a GFRAL protein may be expressed by a recombinant cell genetically modified to express the GFRAL protein on its cell surface. The cell may be present in a composition that includes an isolated GDF15 protein. In certain cases, the cell may additionally express a RET protein, for example the cell may express a RET protein endogenously without being genetically modified to include an exogenous sequence encoding the RET protein. In other embodiments, the cell may not express detectable levels of a RET protein and may be genetically modified to express a RET protein from an exogenous sequence.

Also disclosed herein are fragments of a GFRAL protein, such as GFRAL fragments that lack an intracellular domain present in native GFRAL protein, or the intracellular domain and the transmembrane domain present in native GFRAL protein, such as a native GFRAL depicted in FIG. 1. As noted above, a fragment of a GFRAL protein may also lack a signal sequence present in the native GFRAL and may or may not include a heterologous signal sequence. The fragment may lack the intracellular domain present in a native GFRAL protein but include the transmembrane domain.

The term “GFRAL-extracellular domain” (“GFRAL-ECD”) includes full-length GFRAL ECDs, GFRAL ECD fragments, and GFRAL ECD variants. As used herein, the term “GFRAL ECD” refers to a GFRAL polypeptide with or without a signal peptide that lacks the intracellular and/or transmembrane domains. In some embodiments, a GFRAL ECD refers to a protein having the amino acid sequence that is at least 70% identical to the amino acid sequence of a human full-length GFRAL ECD having the amino acid sequence:

(SEQ ID NO: 1806) QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSY CNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFK WNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNP CDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCA VNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCI STLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQH MLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGE.

The term “full-length GFRAL ECD”, as used herein, refers to a GFRAL ECD that extends to the last amino acid of the extracellular domain, and may or may not include an N-terminal signal peptide. However, it is noted that “full-length GFRAL ECD” also encompasses a GFRAL-ECD that is extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids on the C-terminus to include amino acids residues of the transmembrane domain provided that the polypeptide is soluble. In other words, such a GFRAL ECD lacks a sufficient length of a transmembrane domain such that it is not anchored into a cell membrane. The phrase “full-length GFRAL ECD” also encompasses a GFRAL-ECD that is extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids on the N-terminus to include amino acids residues of the signal peptide. In certain embodiments, a GFRAL ECD refers to a contiguous amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a contiguous amino acid sequence depicted in FIG. 1 and lacks at least 30, 33, 35, 40, 45, 50, or 55 amino acids or more at the C-terminus of the GFRAL sequences depicted in FIG. 1.

A GFRAL ECD is not an ECD of TGFβ RII (Acc. Nos.: NM_001024847.2; NM_003242.5) or orthologs thereof. GFRAL ECD is distinct from ECD of TGFβ RI (Acc. Nos.: NP_001124388.1; NP_004603.1) or orthologs thereof. In certain embodiments, a GFRAL ECD may be a protein having the amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1806.

As used herein, the term “GFRAL ECD fragment” refers to a GFRAL ECD having one or more residues deleted from the N and/or C terminus of the full-length ECD and that retains the ability to bind to GDF15. In some instances, a GFRAL ECD fragment may or may not include an N-terminal signal peptide. In some instances, a GFRAL ECD fragment is a human GFRAL ECD fragment that lacks 1, 5, 10, 15, 16, 17, 18, or 19 residues present at the N-terminus of the sequence:

(SEQ ID NO: 1807) MIVFIFLAMGLSLENEYTSQTNNCTYLREQCLRDANGCKHAWRVMEDACN DSDPGDPCKMRNSSYCNLSIQYLVESNFQFKECLCTDDFYCTVNKLLGKK CINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLAS YLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQ SKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTR KCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSE ESLCKIFQHMLHRKSCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNG E

Another exemplary GFRAL ECD fragment comprises the following amino acid sequence, which corresponds to Q20 to C316 of a full-length human precursor GFRAL protein:

(SEQ ID NO: 1808) QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLS IQYLVESNFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTT RSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQA AIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPT CLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTC SGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSC

Yet another exemplary GFRAL ECD fragment comprises the following amino acid sequence, which corresponds to W115 to E351 of a full-length human precursor GFRAL protein:

(SEQ ID NO: 1809) WNLTTRSHHGFKGMWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDL KQCQAAIRFFYQNIPFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNM VPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSK QDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRK SCFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGE.

The above exemplary GFRAL ECD fragment was used in various methods as described in the Examples, including to produce a crystal of a complex comprising a GFRAL protein and a GDF15 protein or a GFRAL protein and an exemplary anti-GFRAL antibody.

Within a GFRAL protein or GFRAL ECD there are three domains—domain 1 (D1), domain 2 (D2) and domain 3 (D3). In some embodiments, the amino acid sequence of an exemplary D1 domain are residues Q20 to S130 of SEQ ID NO: 1797. In some embodiments, the amino acid sequence of an exemplary D2 domain are residues C131 to C210 of SEQ ID NO: 1797. In some embodiments, the amino acid sequence of an exemplary D3 domain are residues C220 to C316 of SEQ ID NO: 1797. Certain properties of a GFRAL protein can be attributed to the activity and/or binding of these domains, including within the ECD. For example, as described herein, amino acid residues within D2 are identified as being core interaction interface amino acids and/or boundary interaction interface amino acids for a GFRAL protein binding to a GDF15 protein. Likewise, as described herein, amino acid residues within D3 are identified as being core interaction interface amino acids and/or boundary interaction interface amino acids for a GFRAL protein binding to a RET protein.

The term “core interaction interface amino acid” or grammatical equivalent thereof refers to an amino acid residue of a given protein that has at least one atom within less or equal to 4.5 Å from an interacting protein (e.g., an amino acid of a GFRAL protein that interacts with a GDF15 protein or a RET protein). A distance of 4.5 Å allows for atoms within a van der Waals radius plus a possible water-mediated hydrogen bond to form a bond with the interacting protein.

The term “boundary interaction interface amino acid” or grammatical equivalent thereof refers to an amino acid residue of a given protein that has at least one atom within less than or equal to 5 Å from a core interface amino acid on the given protein (e.g., an amino acid of a GFRAL protein that is within 5 Å of a core interaction interface amino acid of a GFRAL protein that interacts with a GDF15 protein or a RET protein). A distance of less than or equal to 5 Å allows proteins binding to residues less than 5 Å away from core interaction interface amino acids on a given protein to be within the van der Waals radius of an interacting protein.

As used herein, the term “GFRAL ECD variants” refers to GFRAL ECDs that contain amino acid additions, deletions, or substitutions and that remain capable of binding to GDF15. Such variants may be at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical to a parent GFRAL ECD.

“Growth differentiation factor 15” or “GDF15,” also known in the art as MIC-1 (macrophage inhibitory cytokine-1), PDF (prostate differentiation factor), PLAB (placental bone morphogenetic protein), NAG-1 (non-steroidal anti-inflammatory drugs (NSAIDs) activated gene), TGF-PL, and PTGFB, is a member of the transforming growth factor β (TGF-β) super-family. GDF15, which is synthesized as a 62 kDa intracellular precursor protein that is subsequently cleaved by a furin-like protease, is secreted as a 25 kDa disulfide-linked protein (see, e.g., Fairlie et al., J. Leukoc. Biol 65:2-5 (1999)). GDF15 mRNA is seen in several tissues, including liver, kidney, pancreas, colon and placenta, and GDF15 expression in liver can be significantly up-regulated during injury of organs such as the liver, kidneys, heart and lungs.

The GDF15 precursor is a 308 amino acid polypeptide (NCBI Ref. Seq. NP_004855.2; GI:153792495) containing a 29 amino acid signal peptide, a 167 amino acid pro-domain, and a mature domain of 112 amino acids which is excised from the pro-domain by furin-like proteases.

An amino acid sequence of a precursor human GDF15 polypeptide is provided below:

(SEQ ID NO: 1810) MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSED SRFRELRKRYEDLLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGH LHLRISRAALPEGLPEASRLHRALFRLSPTASRSWDVTRPLRRQLSLARP QAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAARGRRRARARNG DHCPLGPGRCCRLHTVRASLEDLGWADVVVLSPREVQVTMCIGACPSQFR AANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDD LLAKDCHCI Such a 308-amino acid GDF15 polypeptide is referred to as a “full-length” GDF15 polypeptide; a 112-amino acid GDF15 polypeptide (amino acids 197-308 of “full-length” GDF15) is a “mature” GDF15 polypeptide.

“GDF15” as used herein includes a protein having an amino acid sequence that is at least 65% identical to the amino acid sequence of a mature human GDF15 polypeptide. An amino acid sequence of a mature human GDF15 polypeptide is provided below:

(SEQ ID NO: 1811) ARNGDHCPLGPGRCCRLHTVRASLEDLGWADVVVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQ TYDDLLAKDCHCI

The above exemplary mature human GDF15 was used in the various methods described as in the Examples, including to produce a crystal of a complex comprising a GFRAL protein and a GDF15 protein.

Unless otherwise indicated, the term “GDF15” refers to a 112 amino acid mature human sequence (e.g., SEQ ID NO: 1811). In addition, numerical references to particular GDF15 residues refer to a 112 amino acid mature sequence (e.g., residue 1 is Ala (A), and residue 112 is Ile (I) of SEQ ID NO: 1811). For example, while a GDF15 precursor amino acid sequence predicts three excision sites, resulting in three putative forms of “mature” human GDF15 (e.g., 110, 112 and 115 amino acids), the 112 amino acid mature sequence is accepted as being correct.

Within the context of the present disclosure, “GDF15” or “GDF15 protein” includes GDF15 orthologs, and modified forms thereof, from other mammalian species, and their use, including mouse (NP_035949; GI:170784848), chimpanzee (XP_009433302.1; GI:694973734), orangutan (XP_009251261.1 GI:686757768), Rhesus monkey (EHH29815; GI:355703324), giant panda (XP_002912774; GI:301753921), gibbon (XP_004089328.1; GI:441627981), guinea pig (XP_003465238; GI:348558868), ferret (AER98997; GI:355689945), cow (NP_001193227; GI:329664989), pig (NP_001167527; GI:291291599), dog (XP_541938; GI:57101740) and platypus (Ornithorhynchus anatinus; AFV61279; GI:410111209). Such exemplary GDF15 proteins are shown in FIG. 2, which includes an alignment of the various exemplary GDF15 proteins. A mature form of human GDF15 has approximately 67% amino acid identity to the mouse ortholog.

“RET,” also known in the art as Ret Proto-Oncogene, Cadherin-Related Family Member 16, Rearranged During Transfection, RET Receptor Tyrosine Kinase, Cadherin Family Member 12, Proto-Oncogene C-Ret, EC 2.7.10.1, CDHF12, CDHR16, RET51, PTC, Hydroxyaryl-Protein Kinase, RET Transforming Sequence, and Receptor Tyrosine Kinase, is one of the receptor tyrosine kinases, cell-surface molecules that transduce signals for cell growth and differentiation. RET acts as a co-receptor and is known as a primary signaling receptor for glial-cell-line-derived neurotrophic factor (GDNF) ligands (in human, GDNF, artemin, neurturin, and persephin) when bound to members of the GDNF receptor alpha (GFRα) co-receptors. A RET protein (e.g., a RET-ECD) comprises 4 consecutive cadherin-like domains (CLD1-CLD4) followed by a membrane proximal cystine rich domain (CRD). As disclosed herein, a RET protein is a co-receptor with a GFRAL protein and a GDF15 protein (e.g., acting as a co-receptor with a RET protein). A receptor complex, as described herein, includes a GFRAL protein, such as a RET/GFRAL complex, a GFRAL/GDF15 complex, and a RET/GFRAL/GDF15 complex.

As used herein, “Ret” or “RET” refers to a protein having the amino acid sequence that is at least 75% identical, e.g., 77%, 79%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 1813. RET is distinct from TGFβ RI and TGFβ RII. SEQ ID NO: 1812 is the sequence of a mature human RET9 that lacks a signal peptide:

(SEQ ID NO: 1812) KVALGLYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLY GTYRTRLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYL KVFLSPTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPS FRIRENRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEV STRWALDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFP AGVDTASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDT WAQQTFRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLA VLVNDSDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIG KVCVENCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKAL RRPKCAELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSK RRLECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVV ETQDINICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFPEEEKCFCEPE DIQDPLCDELCRTVIAAAVLFSFIVSVLLSAFCIHCYHKFAHKPPISSAE MTFRRPAQAFPVSYSSSGARRPSLDSMENQVSVDAFKILEDPKWEFPRKN LVLGKTLGEGEFGKVVKATAFHLKGRAGYTTVAVKMLKENASPSELRDLL SEFNVLKQVNHPHVIKLYGACSQDGPLLLIVEYAKYGSLRGFLRESRKVG PGYLGSGGSRNSSSLDHPDERALTMGDLISFAWQISQGMQYLAEMKLVHR DLAARNILVAEGRKMKISDFGLSRDVYEEDSYVKRSQGRIPVKVVMAIES LFDHIYTTQSDVWSFGVLLWEIVTLGGNPYPGIPPERLFNLLKTGHRMER PDNCSEEMYRLMLQCWKQEPDKRPVFADISKDLEKMMVKRRDYLDLAAST PSDSLIYDDGLSEEETPLVDCNNAPLPRALPSTWIENKLYGRISHAFTRF

The amino acid sequence of a full-length precursor human RET protein is provided below, which includes a signal peptide sequence (underlined and lowercase residues):

(SEQ ID NO: 1813) makatsgaaglrlllllllpllgkvalgLYFSRDAYWEKLYVDQAAGTP LLYVHALRDAPEEVPSFRLGQHLYGTYRTRLHENNWICIQEDTGLLYLNR SLDHSSWEKLSVRNRGFPLLTVYLKVFLSPTSLREGECQWPGCARVYFSF FNTSFPACSSLKPRELCFPETRPSFRIRENRPPGTFHQFRLLPVQFLCPN ISVAYRLLEGEGLPFRCAPDSLEVSTRWALDREQREKYELVAVCTVHAGA REEVVMVPFPVTVYDEDDSAPTFPAGVDTASAVVEFKRKEDTVVATLRVF DADVVPASGELVRRYTSTLLPGDTWAQQTFRVEHWPNETSVQANGSFVRA TVHDYRLVLNRNLSISENRTMQLAVLVNDSDFQGPGAGVLLLHFNVSVLP VSLHLPSTYSLSVSRRARRFAQIGKVCVENCQAFSGINVQYKLHSSGANC STLGVVTSAEDTSGILFVNDTKALRRPKCAELHYMVVATDQQTSRQAQAQ LLVTVEGSYVAEEAGCPLSCAVSKRRLECEECGGLGSPTGRCEWRQGDGK GITRNFSTCSPSTKTCPDGHCDVVETQDINICPQDCLRGSIVGGHEPGEP RGIKAGYGTCNCFPEEEKCFCEPEDIQDPLCDELCRTVIAAAVLFSFIVS VLLSAFCIFICYHKFAHKPPISSAEMTFRRPAQAFPVSYSSSGARRPSLD SMENQVSVDAFKILEDPKWEFPRKNLVLGKTLGEGEFGKVVKATAFHLKG RAGYTTVAVKMLKENASPSELRDLLSEFNVLKQVNHPHVIKLYGACSQDG PLLLIVEYAKYGSLRGFLRESRKVGPGYLGSGGSRNSSSLDHPDERALTM GDLISFAWQISQGMQYLAEMKLVHRDLAARNILVAEGRKMKISDFGLSRD VYEEDSYVKRSQGRIPVKWMAIESLFDHIYTTQSDVWSFGVLLWEIVTLG GNPYPGIPPERLFNLLKTGHRMERPDNCSEEMYRLMLQCWKQEPDKRPVF ADISKDLEKMMVKRRDYLDLAASTPSDSLIYDDGLSEEETPLVDCNNAPL PRALPSTWIENKLYGRISHAFTRF

Accordingly, “RET” or a “RET protein” as used herein encompasses human RET and variants thereof, including but not limited to orthologs thereof, such as murine RET, cyno RET, and the like. In certain embodiments, RET may be a protein having the amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1813.

In certain embodiments, an isolated RET-extracellular domain (RET-ECD) polypeptide is provided. A RET-ECD may be bound to a ligand such as a GFRAL protein when present in an isolated protein complex of the present disclosure. The term “RET-extracellular domain” (“RET-ECD”) includes full-length RET ECDs, RET ECD fragments, and RET ECD variants. As used herein, the term “RET ECD” refers to a RET polypeptide with or without a signal peptide that lacks the intracellular and transmembrane domains. In some embodiments, a RET ECD refers to a protein having an amino acid sequence that is at least 75% identical to the amino acid sequence of human full-length RET ECD having the amino acid sequence:

(SEQ ID NO: 1814) KVALGLYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLY GTYRTRLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYL KVFLSPTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPS FRIRENRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEV STRWALDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFP AGVDTASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDT WAQQTFRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLA VLVNDSDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIG KVCVENCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKAL RRPKCAELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSK RRLECEECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVV ETQDINICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFPEEEKCFCEPE DIQDPLCDELCR

In another exemplary embodiment, the a RET ECD refers to a protein having a amino acid sequence that is at least 75% identical to the amino acid sequence of a human full-length RET ECD having the amino acid sequence:

(SEQ ID NO: 1815) LYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLYGTYRT RLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYLKVFLS PTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPSFRIRE NRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEVSTRWA LDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFPAGVDT ASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDTWAQQT FRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLAVLVND SDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIGKVCVE NCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKALRRPKC AELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSKRRLEC EECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDI NICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFPEEEKCFCEPEDIQDP LCDELCR

The term “full-length RET ECD”, as used herein, refers to a RET ECD that extends to the last amino acid of an extracellular domain, and may or may not include an N-terminal signal peptide. However, it is noted that “full-length RET ECD” also encompasses a RET-ECD that is extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids on the C-terminus to include amino acids residues of the transmembrane domain provided that the polypeptide is soluble. In other words, a RET ECD lacks a sufficient length of a transmembrane domain such that it is not anchored into a cell membrane. The phrase “full-length RET ECD” also encompasses a RET-ECD that is extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids on the N-terminus to include amino acids residues of the signal peptide. In certain embodiments, RET fragment refers to a contiguous amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a contiguous amino acid sequence of RET described herein and lacks at least 30, 33, 35, 40, 45, 50, or 55 amino acids or more at the C-terminus of RET sequences described herein.

As used herein, the term “RET ECD fragment” refers to a RET ECD having one or more residues deleted from the N and/or C terminus of a full-length ECD and that retains the ability to bind to a GFRAL protein. In some instances, a RET ECD fragment may or may not include an N-terminal signal peptide. In some instances, a RET ECD fragment is a human RET ECD fragment that lacks 1, 5, 10, 15, 16, 17, 18, or 19 residues present at the N-terminus of the sequence:

(SEQ ID NO: 1816) LYFSRDAYWEKLYVDQAAGTPLLYVHALRDAPEEVPSFRLGQHLYGTYRT RLHENNWICIQEDTGLLYLNRSLDHSSWEKLSVRNRGFPLLTVYLKVFLS PTSLREGECQWPGCARVYFSFFNTSFPACSSLKPRELCFPETRPSFRIRE NRPPGTFHQFRLLPVQFLCPNISVAYRLLEGEGLPFRCAPDSLEVSTRWA LDREQREKYELVAVCTVHAGAREEVVMVPFPVTVYDEDDSAPTFPAGVDT ASAVVEFKRKEDTVVATLRVFDADVVPASGELVRRYTSTLLPGDTWAQQT FRVEHWPNETSVQANGSFVRATVHDYRLVLNRNLSISENRTMQLAVLVND SDFQGPGAGVLLLHFNVSVLPVSLHLPSTYSLSVSRRARRFAQIGKVCVE NCQAFSGINVQYKLHSSGANCSTLGVVTSAEDTSGILFVNDTKALRRPKC AELHYMVVATDQQTSRQAQAQLLVTVEGSYVAEEAGCPLSCAVSKRRLEC EECGGLGSPTGRCEWRQGDGKGITRNFSTCSPSTKTCPDGHCDVVETQDI NICPQDCLRGSIVGGHEPGEPRGIKAGYGTCNCFPEEEKCFCEPEDIQDP LCDELCR 

The above exemplary RET ECD fragment was used in various methods described in the Examples, including to produce a model of a complex comprising a RET protein, a GFRAL protein and a GDF15 protein.

In alternative embodiments of a RET ECD, the RET-ECD comprises a C64R, N75Q, N166Q, or C183S mutation in a RET ECD sequence of human full-length RET ECD SEQ ID NO 1814.

The phrase “modulates the activity and/or signaling,” when applied to a binding protein, such an antibody that binds to GFRAL of the present disclosure, means that the binding protein (e.g., antibody) mimics or modulates an in vitro or an in vivo biological effect induced by the binding of: (i) a GFRAL protein; (ii) a GDF15 protein and a GFRAL protein; or (iii) a GDF15 protein, a GFRAL protein, and a RET protein. In assessing the binding and specificity of anti-GFRAL antibody, for example, an antibody or fragment thereof, that binds to a GFRAL protein (e.g., a human GFRAL protein), is deemed to induce a biological response when the response is equal to or less than 95%, and preferably equal to or less than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, of the activity of a wild type GFRAL standard (e.g., the mature form of a human GFRAL protein). An antibody or fragment thereof, that binds to GFRAL (e.g., human GFRAL), is also deemed to induce a biological response when it has one or more of the following properties: exhibiting an efficacy level of equal to or less than 95% of a GFRAL standard, with an IC50 of equal to or less than 100 nM, e.g., 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 0.1 nM 0.01 nM in (1) a ELK1-luciferase reporter assay (see, e.g., Example 3); or (2) ERK-phosphorylation assay in U2OS cells (see, e.g., Example 4).

The term “binding protein” refers to a protein comprising a portion (e.g., one or more binding regions such as CDRs) that binds to a GFRAL protein, including a human GFRAL protein and, optionally, a scaffold or framework portion (e.g., one or more scaffold or framework regions) that allows the binding portion to adopt a conformation that promotes binding of the binding protein to a GFRAL polypeptide, fragment or epitope. Examples of such binding proteins include antibodies, such as a human antibody, a humanized antibody; a chimeric antibody; a recombinant antibody; a single chain antibody; a diabody; a triabody; a tetrabody; a Fab fragment; a F(ab′) 2 fragment; an IgD antibody; an IgE antibody; an IgM antibody; an IgG1 antibody; an IgG2 antibody; an IgG3 antibody; or an IgG4 antibody, and fragments thereof. The binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, e.g., Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptide antibody mimetics (“PAMs”) can be used, as well as scaffolds based on antibody mimetics utilizing fibronectin components as a scaffold. In the context of the present disclosure, a binding protein is said to specifically bind or selectively bind to GFRAL, for example, when the dissociation constant (K_(D)) is ≤10⁻⁸ M. The binding protein (e.g., antibody) may specifically bind GFRAL with high affinity when the K_(D) is ≤10⁻⁹ M or K_(D) is ≤10⁻¹⁰ M. In some embodiments, the binding proteins (e.g., antibodies) may bind to GFRAL, including with a K_(D) of between about 10⁻⁷ M and about 10⁻¹² M and in other embodiments, the binding proteins (e.g., antibodies) may bind with a K_(D) of 1-2×10⁻⁹ M.

The term “antibody” and “immunoglobulin” or “Ig” are used interchangeably herein, and is used in the broadest sense and specifically covers, for example, individual anti-GFRAL monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), anti-GFRAL antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain anti-GFRAL antibodies, and fragments of anti-GFRAL antibodies, as described below. An antibody can be human, humanized, chimeric and/or affinity matured as well as an antibody from other species, for example mouse, rabbit etc. The term “antibody” is intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (See, Borrebaeck (ed.) (1995) Antibody Engineering, Second Ed., Oxford University Press.; Kuby (1997) Immunology, Third Ed., W.H. Freeman and Company, New York). In specific embodiments, the specific molecular antigen can be bound by an antibody provided herein includes a GFRAL polypeptide, GFRAL fragment or GFRAL epitope. Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments (e.g., antigens-binding fragments such as GFRAL binding fragments) of any of the above, which refers a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non-limiting examples of functional fragments (e.g., antigens-binding fragments such as GFRAL binding fragments) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab′) fragments, F(ab)2 fragments, F(ab′)2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, antigen binding domains or molecules that contain an antigen-binding site that binds to a GFRAL antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-GFRAL antibody). Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. Anti-GFRAL antibodies may be agonistic antibodies or antagonistic antibodies. Antibodies provided herein include antagonistic antibodies to GFRAL, for example, antibodies that inhibit GFRAL signaling. Exemplary anti-GFRAL antibodies include antibodies with CDRs as shown in Tables 1-24.

The terms “about” or “approximately” mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within or 1% or less of a given value or range.

An “antigen” is a predetermined antigen to which an antibody can selectively bind. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide.

The term “antigen binding fragment,” “antigen binding domain,” “antigen binding region,” and similar terms refer to that portion of an antibody which comprises the amino acid residues that interact with an antigen and confer on the binding agent its specificity and affinity for the antigen (e.g., the complementarity determining regions (CDRs)).

The terms “binds” or “binding” refer to an interaction (e.g., covalent or non-covalent) between molecules including, for example, to form a complex. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as GFRAL, is the affinity of the antibody or functional fragment for that epitope. The ratio of association (k1) to dissociation (k−1) of an antibody to a monovalent antigen (k1/k−1) is the association constant K, which is a measure of affinity. The value of K varies for different complexes of antibody and antigen and depends on both k1 and k−1. The association constant K for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent GFRAL, come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity. The avidity of an antibody can be a better measure of its binding capacity than is the affinity of its individual binding sites. For example, high avidity can compensate for low affinity as is sometimes found for pentameric IgM antibodies, which can have a lower affinity than IgG, but the high avidity of IgM, resulting from its multivalence, enables it to bind antigen effectively.

The terms “antibodies that specifically bind to GFRAL,” “antibodies that specifically bind to a GFRAL epitope,” and analogous terms are also used interchangeably herein and refer to antibodies that specifically bind to a GFRAL polypeptide, such as a GFRAL antigen, or fragment, or epitope (e.g., human GFRAL such as a human GFRAL polypeptide, antigen or epitope). An antibody that specifically binds to GFRAL, (e.g., human GFRAL) may bind to the extracellular domain or peptide derived from the extracellular domain of GFRAL. An antibody that specifically binds to a GFRAL antigen (e.g., human GFRAL) may be cross-reactive with related antigens (e.g., cyno GFRAL). In certain embodiments, an antibody that specifically binds to a GFRAL antigen does not cross-react with other antigens. An antibody that specifically binds to a GFRAL antigen can be identified, for example, by immunoassays, Biacore, or other techniques known to those of skill in the art. An antibody binds specifically to a GFRAL antigen when it binds to a GFRAL antigen with higher affinity than to any cross reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme linked immunosorbent assays (ELISAs). Typically a specific or selective reaction will be at least twice background signal or noise and may be more than 10 times background. See, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition, Raven Press, New York at pages 332 336 for a discussion regarding antibody specificity. An antibody “which binds” an antigen of interest (e.g., a target antigen such as GFRAL) is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen, and does not significantly cross-react with other proteins. In such embodiments, the extent of binding of the antibody to a “non-target” protein will be less than about 10% of the binding of the antibody to its particular target protein, for example, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA). With regard to the binding of an antibody to a target molecule, the term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target means binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a Kd for the target of at least about 10⁻⁴ M, alternatively at least about 10⁻⁵ M, alternatively at least about 10⁻⁶ M, alternatively at least about 10⁻⁷ M, alternatively at least about 10⁻⁸ M, alternatively at least about 10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, alternatively at least about 10⁻¹¹ M, alternatively at least about 10⁻¹² M, or greater. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In certain embodiments, an antibody that binds to GFRAL has a dissociation constant (Kd) of less than or equal to 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. The lower the K_(D), the higher the affinity of the anti-GFRAL antibody. In certain embodiments, anti-GFRAL antibody binds to an epitope of GFRAL that is conserved among GFRAL from different species (e.g., between human and cyno GFRAL).

The term “compete” when used in the context of anti-GFRAL antibodies (e.g., antagonistic antibodies and binding proteins that bind to GFRAL) that bind to or compete for the same epitope or binding site on a target means competition between as determined by an assay in which the antibody (or binding fragment) thereof under study prevents or inhibits the specific binding of a reference molecule (e.g., a reference ligand, or reference antigen binding protein, such as a reference antibody) to a common antigen (e.g., GFRAL or a fragment thereof). Numerous types of competitive binding assays can be used to determine if a test antibody competes with a reference antibody for binding to GFRAL (e.g., human GFRAL). Examples of assays that can be employed include solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., (1983) Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., (1988) Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., (1990) Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., (1990) Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of a purified antigen (e.g., GFRAL such as human GFRAL) bound to a solid surface or cells bearing either of an unlabelled test antigen binding protein (e.g., test anti-GFRAL antibody) or a labeled reference antigen binding protein (e.g., reference anti-GFRAL antibody). Competitive inhibition may be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and/or antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference for antibodies steric hindrance to occur. Additional details regarding methods for determining competitive binding and/or binding to the same epitope are described herein. Usually, when a competing antibodies protein is present in excess, it will inhibit specific binding of a reference antibodies to a common antigen by at least 23%, for example 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, 96% or 97%, 98%, 99% or more.

The term “anti-GFRAL antibody” or “an antibody that binds to GFRAL” includes an antibody that is capable of binding GFRAL with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GFRAL. In certain embodiments, the extent of binding of an anti-GFRAL antibody to an unrelated, non-GFRAL protein is less than about 10% of the binding of the antibody to GFRAL as measured, for example, by fluorescence activated cell sorting (FACS) analysis or an immunoassay such as a radioimmunoassay (RIA). An antibody that “specifically binds to” or is “specific for” GFRAL is illustrated herein. In certain embodiments, an antibody that binds to GFRAL, as described herein, has a dissociation constant (Kd) of less than or equal to 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM, and/or is greater than or equal to 0.1 nM. In certain embodiments, anti-GFRAL antibody binds to an epitope of GFRAL that is conserved among GFRAL from different species (e.g., between human and cyno GFRAL).

The terms “crystal”, and “crystallized” as used herein, refer to one or more proteins or fragments thereof that exist in the form of a crystal. Crystals are one form of the solid state of matter, which is distinct from other forms such as the amorphous solid state or the liquid crystalline state. Crystals are composed of regular, repeating, three-dimensional arrays of atoms, ions, molecules (e.g., proteins such as antibodies), or molecular assemblies (e.g., ligand/receptor or antigen/antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well-understood in the field. The fundamental unit, or building block, that is repeated in a crystal is called the asymmetric unit. Repetition of the asymmetric unit in an arrangement that conforms to a given, well-defined crystallographic symmetry provides the “unit cell” of the crystal. Repetition of the unit cell by regular translations in all three dimensions provides the crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999).

An “isolated” antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue source and/or other contaminant components from which the antibody is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of an antibody in which the antibody is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes preparations of antibody having less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of heterologous protein (also referred to herein as a “contaminating protein”). In certain embodiments, when the antibody is recombinantly produced, it is substantially free of culture medium, e.g., culture medium represents less than about 20%, 15%, 10%, 5%, or 1% of the volume of the protein preparation. In certain embodiments, when the antibody is produced by chemical synthesis, it is substantially free of chemical precursors or other chemicals, for example, it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the antibody have less than about 30%, 25%, 20%, 15%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. Contaminant components can also include, but are not limited to, materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method (Lowry et al. J. Bio. Chem. 193: 265-275, 1951), such as 96%, 97%, 98%, or 99%, by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. In specific embodiments, antibodies provided herein are isolated.

A 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for p and E isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6.

The term “variable region” or “variable domain” refers to a portion of the light or heavy chains of an antibody that is generally located at the amino-terminal of the light or heavy chain and has a length of about 120 to 130 amino acids in the heavy chain and about 100 to 110 amino acids in the light chain, and are used in the binding and specificity of each particular antibody for its particular antigen. The variable region of the heavy chain may be referred to as “VH.” The variable region of the light chain may be referred to as “VL.” The term “variable” refers to the fact that certain segments of the variable regions differ extensively in sequence among antibodies. The V region mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable regions. Instead, the V regions consist of less variable (e.g., relatively invariant) stretches called framework regions (FRs) of about 15-30 amino acids separated by shorter regions of greater variability (e.g., extreme variability) called “hypervariable regions” that are each about 9-12 amino acids long. The variable regions of heavy and light chains each comprise four FRs, largely adopting a β sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991)). The constant regions are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). The variable regions differ extensively in sequence between different antibodies. The variability in sequence is concentrated in the CDRs while the less variable portions in the variable region are referred to as framework regions (FR). The CDRs of the light and heavy chains are primarily responsible for the interaction of the antibody with antigen. In specific embodiments, the variable region is a human variable region.

The term “variable region residue numbering as in Kabat” or “amino acid position numbering as in Kabat”, and variations thereof, refers to the numbering system used for heavy chain variable regions or light chain variable regions of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc, according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgG 1 EU antibody. Other numbering systems have been described, including, for example, by AbM, Chothia, Contact, IMGT and AHon. Various numbering systems are illustrated in Tables 1-24.

An “intact” antibody is one comprising an antigen-binding site as well as a light chain constant region CL and at least heavy chain constant regions, CH1, CH2 and CH3. The constant regions may include human constant regions or amino acid sequence variants thereof. Preferably, an intact antibody has one or more effector functions.

“Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include, without limitation, Fab, Fab′, F(ab′)2, and Fv fragments; diabodies and di-diabodies (see, e.g., Holliger, P. et al., (1993) Proc. Natl. Acad. Sci. 90:6444-8; Lu, D. et al., (2005) J. Biol. Chem. 280:19665-72; Hudson et al., Nat. Med. 9:129-134 (2003); WO 93/11161; and U.S. Pat. Nos. 5,837,242 and 6,492,123); single-chain antibody molecules (see, e.g., U.S. Pat. Nos. 4,946,778; 5,260,203; 5,482,858 and 5,476,786); dual variable domain antibodies (see, e.g., U.S. Pat. No. 7,612,181); single variable domain antibodies (SdAbs) (see, e.g., Woolven et al., Immunogenetics 50: 98-101, 1999; Streltsov et al., Proc Natl Acad Sci USA. 101:12444-12449, 2004); and multispecific antibodies formed from antibody fragments.

A “functional fragment” or “binding fragment” or “antigen binding fragment” of a therapeutic antibody will exhibit at least one if not some or all of the biological functions attributed to the intact antibody, the function comprising at least binding to the target antigen, (e.g., a GFRAL binding fragment or fragment that binds to GFRAL).

The term “fusion protein” as used herein refers to a polypeptide that comprises an amino acid sequence of an antibody and an amino acid sequence of a heterologous polypeptide or protein (e.g., a polypeptide or protein not normally a part of the antibody (e.g., a non-anti-GFRAL antigen binding antibody)). The term “fusion” when used in relation to GFRAL or to an anti-GFRAL antibody refers to the joining of a peptide or polypeptide, or fragment, variant and/or derivative thereof, with a heterologous peptide or polypeptide. In certain embodiments, the fusion protein retains the biological activity of the GFRAL or anti-GFRAL antibody. In certain embodiments, the fusion protein comprises a GFRAL antibody VH region, VL region, VH CDR (one, two or three VH CDRs), and/or VL CDR (one, two or three VL CDRs), wherein the fusion protein binds to a GFRAL epitope, a GFRAL fragment and/or a GFRAL polypeptide.

The term “heavy chain” when used in reference to an antibody refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion includes a variable region of about 120 to 130 or more amino acids and a carboxy-terminal portion that includes a constant region (e.g., CH1, CH2, CH3, CH4). The constant region can be one of five distinct types, (e.g., isotypes) referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the heavy chain constant regions. The distinct heavy chains differ in size: α, δ and γ contain approximately 450 amino acids, while p and E contain approximately 550 amino acids. When combined with a light chain, these distinct types of heavy chains give rise to five well known classes (e.g., isotypes) of antibodies, IgA, IgD, IgE, IgG and IgM, respectively, including four subclasses of IgG, namely IgG1, IgG2, IgG3 and IgG4. A heavy chain can be a human heavy chain.

The term “light chain” when used in reference to an antibody refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion includes a variable region of about 100 to about 110 or more amino acids and a carboxy-terminal portion that includes a constant region. The approximate length of a light chain is 211 to 217 amino acids. There are two distinct types, referred to as kappa (κ) of lambda (A) based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. A light chain can be a human light chain.

The term “effective amount” as used herein refers to the amount of a therapy (e.g., an anti-GFRAL antibody or pharmaceutical composition provided herein; see, e.g., antibodies comprising CDR, VH, and/or VL sequences as shown in Tables 1-24) which is sufficient to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with a GDF15-mediated disease, disorder, or condition, including, for example, involuntary body weight loss, a glucose metabolism disorder or a body weight disorder. This term also encompasses an amount necessary for the reduction or amelioration of the advancement or progression of a given GDF15-mediated disease, disorder or condition, reduction or amelioration of the recurrence, development or onset of a GDF15-mediated disease, disorder or condition, and/or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy (e.g., a therapy other than anti-GFRAL antibody provided herein). In some embodiments, the effective amount is administered in one or more doses, including intermittent doses, wherein one ore more doses are given in a treatment period followed by a resting period when an antibody is not administered (e.g., one cycle of treatment period and rest period can be followed with additional cycles, with one or more treatment periods followed by one or more resting periods). In some embodiments, the effective amount of an antibody provided herein is from about 0.1 mg/kg (mg of antibody per kg weight of the subject) to about 100 mg/kg. In certain embodiments, an effective amount of an antibody provided therein is about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, 3 mg/kg, 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, or about 100 mg/kg (or a range therein). In some embodiments, effective amount as used herein also refers to the amount of an antibody provided herein to achieve a specified result (e.g., mimic or modulate an in vitro or an in vivo biological effect induced by the binding of: (i) GFRAL; (ii) GDF15 and GFRAL; or (iii) GDF15, GFRAL, and RET). For example, an effective amount includes an amount (e.g., in one or more doses) of an anti-GFRAL antibody as described herein effective to: (i) increase body weight; (ii) maintain body weight; (iii) reduce body weight loss; (iv) increase body mass (e.g., lean mass or fat mass); (v) maintain body mass (e.g., lean mass or fat mass); or (vi) reduce loss of body mass (e.g., lean mass or fat mass).

The term “host cell” as used herein refers to a particular subject cell that may be transfected with a nucleic acid molecule and the progeny or potential progeny of such a cell. Progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

The term “immunomodulatory agent” and variations thereof including, but not limited to, immunomodulatory agents, as used herein refer to an agent that modulates a host's immune system. In certain embodiments, an immunomodulatory agent used in the combination therapies provided herein does not include an anti-GFRAL antibody or antigen-binding fragment. Immunomodulatory agents include, but are not limited to, small molecules, peptides, polypeptides, proteins, fusion proteins, antibodies, inorganic molecules, mimetic agents, and organic molecules. The term “small molecule” and analogous terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogues, polynucleotides, polynucleotide analogues, nucleotides, nucleotide analogues, organic or inorganic compounds (i.e., including heterorganic and/or ganometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. In some embodiments, an immunomodulatory agent is an immunostimulatory agent. In some embodiments, an immunomodulatory agent is an immunosuppressant agent. In some embodiments, immunomodulatory agents are agents (e.g., antibodies) that modulate (e.g., inhibit or stimulate) proteins known as immune checkpoint molecules (e.g., co-inhibitory or co-stimulatory), for example, C10orf54, CD86, CD80, PDL-1, PDL-2, CTLA-4, PD1, LAG3, BTNL2, B7-H3, B7-H4, a butyrophilin, CD48, CD244, TIM-3, CD200R, CD200, CD160, BTLA, HVEM, LAIR1, TIM1, Galectin 9, TIM3, CD48, 2B4, CD155, CD112, CD113 and TIGIT (e.g., co-inhibitory), and/or CD154, TNFRSF25, GITR, 4-1BB, OX40, CD27, TMIGD2, ICOS, CD28, CD40, TL1A, GITRL, 41BBL, OX40L, CD70, HHLA2, ICOSL, a cytokine, LIGHT, HVEM, CD30, CD30L, B7-H2, CD80, CD86, CD40L, TIM4, TIM1, SLAM, CD48, CD58, CD155, CD112, DR3, GITR, CD2, and CD226 (e.g., co-stimulatory).

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts, and each monoclonal antibody will typically recognize a single epitope on the antigen. In specific embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single hybridoma or other cell, wherein the antibody binds to only a GFRAL epitope as determined, for example, by ELISA or other antigen-binding or competitive binding assay known in the art. The term “monoclonal” is not limited to any particular method for making the antibody. For example, the monoclonal antibodies useful in the present disclosure may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York). Exemplary methods of producing monoclonal antibodies are provided in the Examples herein.

The term “native” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to those which are found in nature and not manipulated, modified, and/or changed (e.g., isolated, purified, selected) by a human being.

The antibodies provided herein can include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

“Humanized” forms of nonhuman (e.g., murine) antibodies are chimeric antibodies that include human immunoglobulins (e.g., recipient antibody) in which the native CDR residues are replaced by residues from the corresponding CDR of a nonhuman species (e.g., donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. A humanized antibody heavy or light chain can comprise substantially all of at least one or more variable regions, in which all or substantially all of the CDRs correspond to those of a nonhuman immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. In certain embodiments, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285-4289 (1992); and U.S. Pat. No. 6,800,738 (issued Oct. 5, 2004), U.S. Pat. No. 6,719,971 (issued Sep. 27, 2005), U.S. Pat. No. 6,639,055 (issued Oct. 28, 2003), U.S. Pat. No. 6,407,213 (issued Jun. 18, 2002), and U.S. Pat. No. 6,054,297 (issued Apr. 25, 2000).

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage-display libraries (Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991) and yeast display libraries (Chao et al., Nature Protocols 1: 755-768 (2006)). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled, e.g., mice (see, e.g., Jakobovits, A., Curr. Opin. Biotechnol. 1995, 6(5):561-6; Bruiggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8(4):455-8; and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.

A “CDR” refers to one of three hypervariable regions (H1, H2 or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of three hypervariable regions (L1, L2 or L3) within the non-framework region of the antibody VL β-sheet framework. Accordingly, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the regions of most hypervariability within the antibody variable (V) domains (Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat, Adv. Prot. Chem. 32:1-75 (1978)). CDR region sequences also have been defined structurally by Chothia as those residues that are not part of the conserved β-sheet framework, and thus are able to adapt different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Both terminologies are well recognized in the art. CDR region sequences have also been defined by AbM, Contact and IMGT. CDR region sequences are illustrated in Tables 1-24. The positions of CDRs within a canonical antibody variable region have been determined by comparison of numerous structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Because the number of residues within a hypervariable region varies in different antibodies, additional residues relative to the canonical positions are conventionally numbered with a, b, c and so forth next to the residue number in the canonical variable region numbering scheme (Al-Lazikani et al., supra (1997)). Such nomenclature is similarly well known to those skilled in the art.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an antibody variable region that are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (see, e.g., Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software (see, e.g., Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions or CDRs are noted below.

Recently, a universal numbering system has been developed and widely adopted, ImMunoGeneTics (IMGT) Information System® (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT is an integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. Herein, the CDRs are referred to in terms of both the amino acid sequence and the location within the light or heavy chain. As the “location” of the CDRs within the structure of the immunoglobulin variable domain is conserved between species and present in structures called loops, by using numbering systems that align variable domain sequences according to structural features, CDR and framework residues and are readily identified. This information can be used in grafting and replacement of CDR residues from immunoglobulins of one species into an acceptor framework from, typically, a human antibody. An additional numbering system (AHon) has been developed by Honegger and Pluckthun, J. Mol. Biol. 309: 657-670 (2001). Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art (see, e.g., Kabat, supra; Chothia and Lesk, supra; Martin, supra; Lefranc et al., supra) and is also illustrated in Tables 1-24. An Exemplary system, shown herein, combines Kabat and Chothia.

Exemplary IMGT Kabat AbM Chothia Contact V_(H) CDR1 26-35 27-38 31-35 26-35 26-32 30-35 V_(H) CDR2 50-65 56-65 50-65 50-58 53-55 47-58 V_(H) CDR3  95-102 105-117  95-102  95-102  96-101  93-101 V_(L) CDR1 24-34 27-38 24-34 24-34 26-32 30-36 V_(L) CDR2 50-56 56-65 50-56 50-56 50-52 46-55 V_(L) CDR3 89-97 105-117 89-97 89-97 91-96 89-96

Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. As used herein, the terms “HVR” and “CDR” are used interchangeably.

The term “constant region” or “constant domain” refers to a carboxy terminal portion of the light and heavy chain which is not directly involved in binding of the antibody to antigen but exhibits various effector function, such as interaction with the Fc receptor. The terms refer to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable region, which contains the antigen binding site. The constant region may contain the CH1, CH2 and CH3 regions of the heavy chain and the CL region of the light chain.

The term “framework” or “FR” residues are those variable region residues flanking the CDRs. FR residues are present, for example, in chimeric, humanized, human, domain antibodies, diabodies, linear antibodies, and bispecific antibodies. FR residues are those variable domain residues other than the hypervariable region residues or CDR residues.

An “affinity matured” antibody is one with one or more alterations (e.g., amino acid sequence variations, including changes, additions and/or deletions) in one or more HVRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For review, see Hudson and Souriau, Nature Medicine 9:129-134 (2003); Hoogenboom, Nature Biotechnol. 23: 1105-1116 (2005); Quiroz and Sinclair, Revista Ingeneria Biomedia 4: 39-51 (2010).

A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds (e.g., GFRAL, as described herein). For example, blocking antibodies or antagonist antibodies may substantially or completely inhibit the biological activity of the antigen (e.g., GFRAL, as described herein).

An “agonist antibody” is an antibody that triggers a response, e.g., one that mimics at least one of the functional activities of a polypeptide of interest. An agonist antibody includes an antibody that is a ligand mimetic, for example, wherein a ligand binds to a cell surface receptor and the binding induces cell signaling or activities via an intercellular cell signaling pathway and wherein the antibody induces a similar cell signaling or activation.

An “antagonist” of GFRAL refers to a molecule (e.g., antibody) that is capable of detectably inhibiting or otherwise decreasing one or more of the biological activities of GFRAL, such as in a cell expressing GFRAL. In some embodiments, an antagonist of GFRAL (e.g., an agonistic antibody as described herein) may, for example, act by detectably inhibiting or otherwise decreasing the activation and/or cell signaling pathways of a cell expressing a GFRAL, thereby detectably decreasing a GFRAL-mediated biological activity of the cell relative to the GFRAL-mediated biological activity in the absence of antagonist. In some embodiments the antibodies provided herein are antagonistic anti-GFRAL antibodies, including antibodies that inhibit signaling of a complex comprising GFRAL, GDF15 and/or RET. The inhibition or decrease caused by a GFRAL antagonist need not be complete as long as it is detectable using an assay. For example, a cell-based assay described in the Examples below can be used to analyze a biological activity of GFRAL.

“Binding affinity” generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a binding protein such as an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be represented by the dissociation constant (K_(D)). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following. In one embodiment, the “K_(D)” or “K_(D) value” may be measured by assays known in the art, for example by a binding assay. The K_(D) may be measured in a radiolabeled antigen binding assay (RIA), for example, performed with the Fab version of an antibody of interest and its antigen (Chen, et al., (1999) J. Mol Biol 293:865-881). The K_(D) or K_(D) value may also be measured by using surface plasmon resonance assays by Biacore, using, for example, a BIAcore™-2000 or a BIAcore™-3000 BIAcore, Inc., Piscataway, N.J.), or by biolayer interferometry using, for example, the OctetQK384 sytem (ForteBio, Menlo Park, Calif.). An “on-rate” or “rate of association” or “association rate” or “kon” may can also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above using, for example, a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.), or the OctetQK384 sytem (ForteBio, Menlo Park, Calif.).

The phrase “substantially similar” or “substantially the same” denotes a sufficiently high degree of similarity between two numeric values (e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by the values (e.g., K_(D) values). For example, the difference between the two values may be less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, as a function of the value for the reference antibody.

The phrase “substantially reduced,” or “substantially different”, as used herein, denotes a sufficiently high degree of difference between two numeric values (e.g., one associated with an antibody of the present disclosure and the other associated with a reference antibody) such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by the values. For example, the difference between said two values may be preferably greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50% as a function of the value for the reference antibody.

Antibody “effector functions” refer to those biological activities attributable to the Fc region (e.g., a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain, including, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is often defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

A “functional Fc region” possesses an “effector function” of a native sequence Fc region. Exemplary “effector functions” include C1q binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding region or binding domain (e.g., an antibody variable region or domain) and can be assessed, including using various assays as disclosed herein and/or as known in the art.

A “native sequence Fc region” comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature, and not manipulated, modified, and/or changed (e.g., isolated, purified, selected, including or combining with other sequences such as variable region sequences) by a human. Native sequence human Fc regions include a native sequence human IgG1 Fc region (non-A and A allotypes); native sequence human IgG2 Fc region; native sequence human IgG3 Fc region; and native sequence human IgG4 Fc region as well as naturally occurring variants thereof.

A “variant Fc region” comprises an amino acid sequence which differs from that of a native sequence Fc region by virtue of at least one amino acid modification, (e.g., substituting, addition, or deletion) preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the parent polypeptide. The variant Fc region herein will preferably possess at least about 80% homology with a native sequence Fc region and/or with an Fc region of a parent polypeptide, and more preferably at least about 90% homology therewith, for example, at least about 95% homology therewith. For example, a variant with two amino acid changes to alanine at two positions in the human IgG1 Fc sequence are shown bolded in the amino acid sequence provided below:

(SEQ ID NO: 2001) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

For example, a variant with two amino acid changes to alanine at two positions in a truncated human IgG1 Fc sequence, in which the C-terminal lysine residue is absent (IgG1ΔK Fc), are shown bolded in the amino acid sequence provided below:

(SEQ ID NO: 2002) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

For example, a truncated variant of the human IgG1 Fc sequence, in which the C-terminal lysine residue is absent (IgG1ΔK Fc) is shown in the amino acid sequence provided below:

(SEQ ID NO: 2003) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

For example, a variant with an amino acid change to proline at a position in the human IgG4 Fc sequence is shown bolded in the amino acid sequence provided below:

(SEQ ID NO: 2004) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNVVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK

For example, a variant with an amino acid change to proline at a position in a human IgG4 Fc sequence, in which the C-terminal lysine is absent (IgG4 AK Fc), is shown bolded in the amino acid sequence provided below:

(SEQ ID NO: 2005) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQED PEVQFNVVYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLG

For example, a variant with an amino acid change to Glutamine at a position in the human IgG1 Fc sequence is shown bolded in the amino acid sequence provided below:

(SEQ ID NO: 2006) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Any of the VH domains of Tables 1-24 and of FIGS. 4A, 5A, 5C, 5E, 6A, 7A, 8A, 9A, and 10A may be combined with a variant Fc region described herein. Exemplary heavy chain constructs comprising a variant Fc region may include the following constructs designated as shown below; the variable region sequence is bolded with CDR sequences underlined:

3P10 Fab Hc: (SEQ ID NO: 1824) QIQLVQSGPELKKPGETVKISCKAS GYTFTDYGVI WVKQAPGKALKWMG W INTYTGEPTYADDLKG RFAFSLETSASSASLQINNLKNEDTATYFCAR RY GPEDIDY WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDEVD 25M22 Fab Hc: (SEQ ID NO: 1826) QVQLQQSGPDLVKPGASVKISCKAS GYTFTSYWVN WMKQRPGKGLEWIG R IYPGDGDTNYNGKFKG KATLTADKSSSTAYMQLSSLTSEDSAVYFCAR AY LLRLRRTGYYAMDY WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAAL GCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSS LGTQTYICNVNHKPSNTKVDKKVEPKSCDEVD 8D8 Fab Hc: (SEQ ID NO: 1828) QVQLKESGPGLVAPSQSLSITCTVS GFSLSRYSVH WVRQPPGKGLEWLG M IWGFGSTDYNSALKS RLSITKDNSKSQFFLKMNSLQTDDTAMYYCAR IHT TAGSY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKKVEPKSCDEVDG 5F12 Fab Hc: (SEQ ID NO: 1830) QVQLKQSGTELVRPGASVKLSCKAS GYTFTDYYIN WVKQRPGQGLEWIA R IYPGNGNTYHNEKFKG KATLTAEKSSSTAYMQLSSLTSEDSAVYFCAR EG LYYDYDRYFDY WGQGTALTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDKKVEPKSCDEVDG

A “light chain constant region” includes kappa and lambda constant regions. Any of the VL domains of Tables 1-24 and of FIGS. 4B, 5B, 5D, 5F, 6B, 7B, 8B, 9B, and 10B may be combined with a kappa or lambda constant region described herein.

An exemplary kappa constant region is provided below:

(SEQ ID NO: 2007) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

An exemplary lambda constant region is provided below:

(SEQ ID NO: 2008) GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVK AGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECS

Exemplary light chain constructs comprising a constant region may include the following constructs designated as shown below; the variable region sequence is bolded with CDR sequences underlined:

3P10 Fab Lc: (SEQ ID NO: 1825) DIVLTQSPVSLAVSLGQRATISC RASESVDNYGISFMS WFQQKPGQPPKL LIYAASHQGSGVPARFSGSGSGTDFSLNIHPMEEDDSAMYFC LQSKEVPW T FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC 25M22 Fab Lc: (SEQ ID NO: 1827) DVVLTQTPLSLPVNIGDQASISC KSTKSLLNSDEFTYLD WYLQKPGQSPQ LLIF LVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQSNYLP YT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC 8D8 Fab Lc: (SEQ ID NO: 1829) DIVMTQSQKFMSTSIGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALVYS TSYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCHQYNSYPLTFGA GTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 5F12 Fab Lc: (SEQ ID NO: 1831) NIVLTQSPASLAVSLGQRATISCRASESVDTYGNSFMHWYQQKPGQPPKL LIY LASNLES GVPARFSGSGSRTDFTLTIDPVEADDAATYYC HQNNEDPP AFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

The term “variant” when used in relation to GFRAL or to an anti-GFRAL antibody may refer to a peptide or polypeptide comprising one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) amino acid sequence substitutions, deletions, and/or additions as compared to a native or unmodified GFRAL sequence. For example, a GFRAL variant may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native GFRAL. Also by way of example, a variant of an anti-GFRAL antibody may result from one or more (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5) changes to an amino acid sequence of a native or previously unmodified anti-GFRAL antibody. Variants may be naturally occurring, such as allelic or splice variants, or may be artificially constructed. Polypeptide variants may be prepared from the corresponding nucleic acid molecules encoding the variants. In some embodiments, the GFRAL variant or anti-GFRAL antibody variant at least retains GFRAL or anti-GFRAL antibody functional activity, respectively. In some embodiments, an anti-GFRAL antibody variant binds GFRAL and/or is antagonistic to GFRAL activity. In some embodiments, an anti-GFRAL antibody variant binds GFRAL and/or is agonistic to GFRAL activity. In some embodiments, the variant is encoded by a single nucleotide polymorphism (SNP) variant of a nucleic acid molecule that encodes GFRAL or anti-GFRAL antibody VH or VL regions or subregions, such as one or more CDRs.

The term “vector” refers to a substance that is used to carry or include a nucleic acid sequences, including for example, in order to introduce a nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell's chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g. both an antibody heavy and light chain or an antibody VH and VL) both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g. an anti-GFRAL antibody as described herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigen-bearing target cell and subsequently kill the target cell with cytotoxins. The antibodies “arm” the cytotoxic cells and are absolutely required for such killing. The primary cells for mediating ADCC, NK cells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is known (see, e.g., Table 3, page 464, Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991)). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, (see, e.g., U.S. Pat. No. 5,500,362 or 5,821,337) may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in a animal model (see, e.g., Clynes et al. (USA) 95:652-656 (1998)). Antibodies with little or no ADCC activity may be selected for use.

“Fc receptor” or “FcR” describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one that binds an IgG antibody (e.g., a gamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof (see, e.g., review Dasron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are known (see, e.g., Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995)). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (see, e.g., Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). Antibody variants with improved or diminished binding to FcRs have been described (see, e.g., in WO 2000/42072; U.S. Pat. Nos. 7,183,387, 7,332,581 and 7.335,742; Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001)).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) which are bound to their cognate antigen. To assess complement activation, a CDC assay, (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996)), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with a variant Fc region) and increased or decreased C1q binding capability have been described, (see, e.g., U.S. Pat. No. 6,194,551, WO 1999/51642, Idusogie et al. J. Immunol. 164: 4178-4184 (2000)). Antibodies with little or no CDC activity may be selected for use.

In calculating percent identity, the sequences being compared may be aligned in a way that gives the largest match between the sequences. Computer program may be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., (1984) Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences may be aligned for optimal matching of their respective amino acid or nucleotide (the “matched span”, as determined by the algorithm). A gap opening penalty (which is calculated as 3.times. the average diagonal, wherein the “average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 1/10 times the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm. In certain embodiments, a standard comparison matrix (see, Dayhoff et al., (1978) Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.

Exemplary parameters for determining percent identity for polypeptides or nucleotide sequences using the GAP program are the following: (i) Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453; (ii) Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra; (iii) Gap Penalty: 12 (but with no penalty for end gaps) (iv) Gap Length Penalty: 4; and (v) Threshold of Similarity: 0.

Certain alignment schemes for aligning two amino acid sequences may result in matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, the selected alignment method (e.g., the GAP program) can be adjusted if so desired to result in an alignment that spans a number of amino acids, for example, at least 50 contiguous amino acids of the target polypeptide.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

A “modification” of an amino acid residue/position refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/positions. For example, typical modifications include substitution of the residue with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (e.g., generally fewer than 5, 4 or 3) amino acids adjacent to said residue/position, and/or deletion of said residue/position.

An “epitope” is the site on the surface of an antigen molecule to which a single antibody molecule binds, such as a localized region on the surface of an antigen, such as a GFRAL polypeptide, a GFRAL polypeptide fragment or a GFRAL epitope, that is capable of being bound to one or more antigen binding regions of an antibody, and that has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human), that is capable of eliciting an immune response. An epitope having immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a polypeptide to which an antibody binds as determined by any method well known in the art, including, for example, by an immunoassay. Antigenic epitopes need not necessarily be immunogenic. Epitopes often consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics. The term, “epitope” specifically includes linear epitopes and conformational epitopes. A region of a polypeptide contributing to an epitope may be contiguous amino acids of the polypeptide or the epitope may come together from two or more non-contiguous regions of the polypeptide. The epitope may or may not be a three-dimensional surface feature of the antigen. In certain embodiments, a GFRAL epitope is a three-dimensional surface feature of a GFRAL polypeptide. In other embodiments, a GFRAL epitope is linear feature of a GFRAL polypeptide. Generally an antigen has several or many different epitopes and may react with many different antibodies.

An antibody binds “an epitope” or “essentially the same epitope” or “the same epitope” as a reference antibody, when the two antibodies recognize identical, overlapping or adjacent epitopes in a three-dimensional space. The most widely used and rapid methods for determining whether two antibodies bind to identical, overlapping or adjacent epitopes in a three-dimensional space are competition assays, which can be configured in a number of different formats, for example, using either labeled antigen or labeled antibody. In some assays, the antigen is immobilized on a 96-well plate, or expressed on a cell surface, and the ability of unlabeled antibodies to block the binding of labeled antibodies is measured using radioactive, fluorescent or enzyme labels.

“Epitope mapping” is the process of identifying the binding sites, or epitopes, of antibodies on their target antigens. Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure. Induced epitopes are formed when the three dimensional structure of the protein is in an altered confirmation, such as following activation or binding of another protein or ligand.

“Epitope binning” is the process of grouping antibodies based on the epitopes they recognize. More particularly, epitope binning comprises methods and systems for discriminating the epitope recognition properties of different antibodies, using competition assays combined with computational processes for clustering antibodies based on their epitope recognition properties and identifying antibodies having distinct binding specificities.

A “GFRAL-mediated disease,” “GFRAL-mediated disorder,” and “GFRAL-mediated condition” are used interchangeably and refer to any disease, disorder or condition that is completely or partially caused by or is the result of GFRAL or the interaction of a GFRAL with GDF15 and/or alternatively any disease, disorder, or condition in which it is desirable to inhibit the in vivo effects of GDF15. GFRAL-mediated diseases, disorders, or conditions include GDF15-mediated diseases disorders or conditions.

A “GDF15-mediated disease,” “GDF15-mediated disorder,” and “GDF15-mediated condition” are used interchangeably and refer to any disease, disorder or condition that is: (i) completely or partially caused by; or (ii) is the result of a GDF15 protein (e.g., an activity of a GDF15 protein, such as GDF15 signaling or elevated levels of a GDF15 protein) or the interaction of a GFRAL protein with a GDF15 protein and/or a RET protein, alternatively any disease, disorder, or condition in which it is desirable to inhibit the in vivo effects of GDF15. GDF15-mediated diseases, disorders, or conditions include involuntary weight loss, cachexia, sarcopenia, muscle wasting, bone wasting, a cardiovascular disease, a chronic inflammatory disease (e.g., chronic renal disease, chronic obstructive pulmonary disease), and a cancer, including a cancer that has decreased sensitivity to (e.g., resistance to) a chemotherapeutic agent (e.g., an anti-tumor antibody such as trastuzumab) that is induced by or related to a GDF15 protein.

The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., an antibody described herein or any other agent described herein) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder or condition, and/or a symptom related thereto. A therapeutically effective amount of a agent, including a therapeutic agent, can be an amount necessary for (i) reduction or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and/or (iii) to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an antibody provided herein). A “therapeutically effective amount” of a substance/molecule/agent of the present disclosure (e.g., an anti-GFRAL antibody) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule/agent, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule/agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term “therapeutically effective amount” refers to an amount of an antibody or other agent (e.g., or drug) effective to “treat” a disease, disorder, or condition, in a subject or mammal.

A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of a disease, disorder, or condition, a prophylactically effective amount may be less than a therapeutically effective amount.

“Chronic” administration refers to administration of the agent(s) in a continuous mode (e.g., for a period of time such as days, weeks, months or years) as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

Administration “in combination with” one or more further agents includes simultaneous (e.g., concurrent) and consecutive administration in any order. The term “in combination” in the context of the administration of other therapies (e.g., other agents) includes the use of more than one therapy (e.g., one agent). The use of the term “in combination” does not restrict the order in which therapies are administered to a subject. A first therapy (e.g., agent) can be administered before (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks), concurrently, or after (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks) the administration of a second therapy (e.g., agent) to a subject which had, has, or is susceptible to or has a risk of a GDF15-mediated disease, disorder or condition.

Any additional therapy (e.g., agent) can be administered in any order with the other additional therapies (e.g., agents). In certain embodiments, the antibodies can be administered in combination with one or more therapies such as agents (e.g., therapies, including agents, that are not the antibodies that are currently administered) to prevent, treat, manage, and/or ameliorate a GDF15-mediated disease, disorder or condition, or a symptom thereof. Non-limiting examples of therapies (e.g., agents) that can be administered in combination with an antibody include, for example, analgesic agents, anesthetic agents, antibiotics, or immunomodulatory agents or any other agent listed in the U.S. Pharmacopoeia and/or Physician's Desk Reference. Examples of agents useful in combination therapy include, but are not limited to, the following: non-steroidal anti-inflammatory drug (NSAID) such as aspirin, ibuprofen, and other propionic acid derivatives (alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen), acetic acid derivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, fuirofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac), fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid), biphenylcarboxylic acid derivatives (diflunisal and flufenisal), oxicams (isoxicam, piroxicam, sudoxicam and tenoxican), salicylates (acetyl salicylic acid, sulfasalazine) and the pyrazolones (apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone, phenylbutazone). Other combinations include cyclooxygenase-2 (COX-2) inhibitors. Other agents for combination include steroids such as prednisolone, prednisone, methylprednisolone, betamethasone, dexamethasone, or hydrocortisone. Such a combination may be especially advantageous, since one or more side-effects of the steroid can be reduced or even eliminated by tapering the steroid dose required when treating subjects in combination with the present antibodies. Additional examples of agents for combinations include cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists of other human cytokines or growth factors, for example, TNF, LT, IL-1p, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, or PDGF. Combinations of agents may include TNF antagonists like chimeric, humanized or human TNF antibodies, REMICADE, anti-TNF antibody fragments (e.g., CDP870), and soluble p55 or p75 TNF receptors, derivatives thereof, p75TNFRIgG (ENBREL®) or p55TNFR1gG (LENERCEPT®), soluble IL-13 receptor (sIL-13), and also TNFα converting enzyme (TACE) inhibitors; similarly IL-1 inhibitors (e.g., Interleukin-1-converting enzyme inhibitors) may be effective. Other combinations include Interleukin 11, anti-P7s and p-selectin glycoprotein ligand (PSGL). Other examples of agents useful in combination therapy include interferon-β1a (AVONEX); interferon-β1b (BETASERON®); copaxone; hyperbaric oxygen; intravenous immunoglobulin; clabribine; and antibodies to or antagonists of other human cytokines or growth factors (e.g., antibodies to CD40 ligand and CD80).

“Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight ((e.g., less than about 10 amino acid residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™. The term “carrier” can also refer to a diluent, adjuvant (e.g., Freund's adjuvant (complete or incomplete)), excipient, or vehicle with which the therapeutic is administered. Such carriers, including pharmaceutical carriers, can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a exemplary carrier when a composition (e.g., a pharmaceutical composition) is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients (e.g., pharmaceutical excipients) include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral compositions, including formulations, can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa. Compositions, including pharmaceutical compounds, may contain a prophylactically or therapeutically effective amount of an anti-GFRAL antibody, for example, in isolated or purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject (e.g., patient). The formulation should suit the mode of administration.

The term “pharmaceutically acceptable” as used herein means being approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized Pharmacopeia for use in animals, and more particularly in humans.

The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient (e.g., an anti-GFRAL antibody) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation may be sterile.

A “sterile” formulation is aseptic or free from all living microorganisms and their spores.

“Polyclonal antibodies” as used herein refers to an antibody population generated in an immunogenic response to a protein having many epitopes and thus includes a variety of different antibodies directed to the same and to different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (See, e.g., Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York).

An “isolated nucleic acid” is a nucleic acid, for example, an RNA, DNA, or a mixed polymer, which is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. An “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In a specific embodiment, one or more nucleic acid molecules encoding an antibody as described herein are isolated or purified. The term embraces nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems. A substantially pure molecule may include isolated forms of the molecule.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. “Oligonucleotide,” as used herein, generally refers to short, generally single-stranded, generally synthetic polynucleotides that are generally, but not necessarily, less than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for polynucleotides is equally and fully applicable to oligonucleotides. A cell that produces an anti-GFRAL antibody of the present disclosure may include a parent hybridoma cell, as well as bacterial and eukaryotic host cells into which nucleic acid encoding the antibodies have been introduced. Suitable host cells are disclosed below.

As used herein, the terms “treat,” “treatment” and “treating” refer to the reduction or amelioration of the progression, severity, and/or duration of a GDF15-mediated disease, disorder or condition resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as an antibody provided herein). In some embodiments, the agent is an anti-GFRAL antibody. Treatment as used herein includes, but is not limited to, (i) increase body weight; (ii) maintain body weight; (iii) reduce body weight loss; (iv) increase body mass (e.g., lean mass or fat mass); (v) maintain body mass (e.g., lean mass or fat mass); (vi) reduce loss of body mass (e.g., lean mass or fat mass), or any combination thereof.

As used herein, the term “prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a GDF15-mediated disease, disorder or condition and/or symptom related thereto in a subject. In some embodiments, the term “prophylactic agent” refers to an antibody provided herein. In some embodiments, the term “prophylactic agent” refers to an agent other than an antibody provided herein. In some embodiments, a prophylactic agent is an agent which is known to be useful to or has been or is currently being used to prevent a GDF15-mediated disease, disorder or condition and/or a symptom related thereto or impede the onset, development, progression and/or severity of a GDF15-mediated disease, disorder or condition and/or a symptom related thereto. In some embodiments, the prophylactic agent is a fully human anti-GFRAL antibody, such as a fully human anti-GFRAL monoclonal antibody.

The term “prophylactic agent” refers to any agent that can totally or partially inhibit the development, recurrence, onset or spread of a GDF15-mediated disease, disorder or condition, or a symptom thereof in a subject. In certain embodiments, the term “prophylactic agent” refers to an anti-GFRAL antibody as described herein. In certain other embodiments, the term “prophylactic agent” refers to an agent other than an anti-GFRAL antibody as described herein. In certain embodiments, a prophylactic agent is an agent which is known to be useful to or has been or is currently being used to prevent a GDF15-mediated disease, disorder or condition, or a symptom thereof or impede the onset, development, progression and/or severity of a GDF15-mediated disease, disorder or condition, or a symptom thereof. In specific embodiments, the prophylactic agent is a humanized anti-GFRAL antibody, such as a humanized anti-GFRAL monoclonal antibody.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.

The terms “prevent,” “preventing,” and “prevention” refer to the total or partial inhibition of the development, recurrence, onset or spread of a GDF15-mediated disease, disorder or condition, or a symptom thereof, resulting from the administration of a therapy or combination of therapies provided herein (e.g., a combination of prophylactic or therapeutic agents, such as an antibody provided herein).

The term “recombinant antibody” refers to an antibody that is prepared, expressed, created or isolated by recombinant means. Recombinant antibodies can be antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse or cow) that is transgenic and/or transchromosomal for human immunoglobulin genes (see, e.g., Taylor, L. D. et al. (1992) Nucl. Acids Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies can have variable and constant regions, including those derived from human germline immunoglobulin sequences (See Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, however, such recombinant antibodies may be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

The term “side effects” encompasses unwanted and adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). Unwanted effects are not necessarily adverse. An adverse effect from a therapy (e.g., a prophylactic or therapeutic agent) might be harmful or uncomfortable or risky. Examples of side effects include, diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramping, fever, pain, loss of body weight, dehydration, alopecia, dyspenea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, and loss of appetite, rashes or swellings at the site of administration, flu-like symptoms such as fever, chills and fatigue, digestive tract problems and allergic reactions. Additional undesired effects experienced by patients are numerous and known in the art. Many are described in the Physician's Desk Reference (60^(th) ed., 2006).

The terms “subject” and “patient” are used interchangeably herein and, in the context of the methods disclosed herein, refer to an animal that is the recipient of a therapy or preventive case. As used herein, in certain embodiments, a subject is a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkey and human). In specific embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having a GDF15-mediated disease, disorder or condition. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing a GDF15-mediated disease, disorder or condition.

“Substantially all” refers to refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

The term “therapeutic agent” refers to any agent that can be used in treating, preventing or alleviating a disease, disorder or condition, including in the treatment, prevention or alleviation of one or more symptoms of a GDF15-mediated disease, disorder or condition, or a symptom thereof. In certain embodiments, a therapeutic agent refers to an anti-GFRAL antibody as described herein. In certain other embodiments, a therapeutic agent refers to an agent other than an antibody provided herein. In certain embodiments, a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the treatment, prevention or alleviation of one or more symptoms of a GDF15-mediated disease, disorder or condition, or a symptom thereof.

The combination of therapies (e.g., use of prophylactic or therapeutic agents) which is more effective than the additive effects of any two or more single therapy. For example, a synergistic effect of a combination of prophylactic and/or therapeutic agents permits the use of lower dosages of one or more of the agents and/or less frequent administration of the agents to a subject with a GDF15-mediated disease, disorder or condition. The ability to utilize lower dosages of prophylactic or therapeutic therapies and/or to administer the therapies less frequently reduces the toxicity associated with the administration of the therapies to a subject without reducing the efficacy of the therapies in the prevention, management, treatment or amelioration of a GDF15-mediated disease, disorder or condition. In addition, a synergistic effect can result in improved efficacy of therapies in the prevention, or in the management, treatment or amelioration of a GDF15-mediated disease, disorder or condition. Finally, synergistic effect of a combination of therapies (e.g., prophylactic or therapeutic agents) can avoid or reduce adverse or unwanted side effects associated with the use of any single therapy. In some embodiments, the combination therapy comprises an antibody provided herein and insulin (e.g., insulin supplementation). In some embodiments, the combination therapy comprises an-anti-GFRAL antibody and insulin, wherein the combination therapy comprises insulin at a lower daily dosage than the normal daily dosage in an insulin-only therapy. In some embodiments, the combination therapy comprises, e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, or less than 1% of the normal daily insulin dosage in an insulin-only therapy.

The term “therapy” refers to any protocol, method and/or agent that can be used in the prevention, management, treatment and/or amelioration of a GDF15-mediated disease, disorder or condition (e.g., type 1 diabetes or type 2 diabetes). In some embodiments, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and/or other therapies useful in the prevention, management, treatment and/or amelioration of a GDF15-mediated disease, disorder or condition known to one of skill in the art such as medical personnel.

The term “detectable probe” refers to a composition that provides a detectable signal. The term includes, without limitation, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, and the like, that provide a detectable signal via its activity.

The term “diagnostic agent” refers to a substance administered to a subject that aids in the diagnosis of a disease, disorder, or conditions. Such substances can be used to reveal, pinpoint, and/or define the localization of a disease causing process. In certain embodiments, a diagnostic agent includes a substance that is conjugated to an anti-GFRAL antibody as described herein, that when administered to a subject or contacted to a sample from a subject aids in the diagnosis a GDF15-mediated disease, disorder or condition.

The term “detectable agent” refers to a substance that can be used to ascertain the existence or presence of a desired molecule, such as an anti-GFRAL antibody as described herein, in a sample or subject. A detectable agent can be a substance that is capable of being visualized or a substance that is otherwise able to be determined and/or measured (e.g., by quantitation).

The term “encode” or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.

The term “excipient” refers to an inert substance which is commonly used as a diluent, vehicle, preservative, binder, or stabilizing agent, and includes, but not limited to, proteins (e.g., serum albumin, etc.), amino acids (e.g., aspartic acid, glutamic acid, lysine, arginine, glycine, histidine, etc.), fatty acids and phospholipids (e.g., alkyl sulfonates, caprylate, etc.), surfactants (e.g., SDS, polysorbate, nonionic surfactant, etc.), saccharides (e.g., sucrose, maltose, trehalose, etc.) and polyols (e.g., mannitol, sorbitol, etc.). See, also, Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa., which is hereby incorporated by reference in its entirety.

In the context of a peptide or polypeptide, the term “fragment” as used herein refers to a peptide or polypeptide that comprises less than the full length amino acid sequence. Such a fragment may arise, for example, from a truncation at the amino terminus, a truncation at the carboxy terminus, and/or an internal deletion of a residue(s) from the amino acid sequence. Fragments may, for example, result from alternative RNA splicing or from in vivo protease activity. In certain embodiments, fragments include polypeptides comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least contiguous 100 amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 800, at least 850, at least 900, or at least 950, contiguous amino acid residues of the amino acid sequence of a GFRAL polypeptide or an antibody that binds to a GFRAL polypeptide. In some embodiments, a fragment of an antibody that binds to a GFRAL polypeptide retains at least 1, at least 2, or at least 3 or more functions of the antibody.

The terms “manage,” “managing,” and “management” refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent), which does not result in a cure of the disease. In certain embodiments, a subject is administered one or more therapies (e.g., prophylactic or therapeutic agents, such as an antibody provided herein) to “manage” a GDF15-mediated disease, disorder or condition, or a symptom thereof, so as to prevent the progression or worsening of the disease.

“Administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an anti-GFRAL antibody as described herein) into a subject, such as by mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, disorder, or condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease, disorder, or condition, or symptoms thereof. When a disease, disorder, or condition or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease, disorder, or condition, or symptoms thereof.

In the context of a polypeptide, the term “analog” as used herein refers to a polypeptide that possesses a similar or identical function as a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or an anti-GFRAL antibody but does not necessarily comprise a similar or identical amino acid sequence of a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or an anti-GFRAL antibody, or possess a similar or identical structure of a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or an anti-GFRAL antibody. A polypeptide that has a similar amino acid sequence refers to a polypeptide that satisfies at least one of the following: (a) a polypeptide having an amino acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a GFRAL polypeptide (e.g., SEQ ID NO:500, a fragment of a GFRAL polypeptide, or an anti-GFRAL antibody described herein; (b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or an anti-GFRAL antibody (or VH or VL region thereof) described herein of at least 5 amino acid residues, at least 10 amino acid residues, at least 15 amino acid residues, at least 20 amino acid residues, at least 25 amino acid residues, at least 40 amino acid residues, at least 50 amino acid residues, at least 60 amino residues, at least 70 amino acid residues, at least 80 amino acid residues, at least 90 amino acid residues, at least 100 amino acid residues, at least 125 amino acid residues, or at least 150 amino acid residues (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.); and (c) a polypeptide encoded by a nucleotide sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the nucleotide sequence encoding a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or an anti-GFRAL antibody (or VH or VL region thereof) described herein. A polypeptide with similar structure to a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or an anti-GFRAL antibody described herein refers to a polypeptide that has a similar secondary, tertiary or quaternary structure of a GFRAL polypeptide, a fragment of a GFRAL, or a GFRAL antibody described herein. The structure of a polypeptide can determined by methods known to those skilled in the art, including but not limited to, X-ray crystallography, nuclear magnetic resonance, and crystallographic electron microscopy.

The term “composition” is intended to encompass a product containing the specified ingredients (e.g., an antibody provided herein) in, optionally, the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in, optionally, the specified amounts.

In the context of a polypeptide, the term “derivative” as used herein refers to a polypeptide that comprises an amino acid sequence of a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or an antibody that binds to a GFRAL polypeptide which has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term “derivative” as used herein also refers to a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or an antibody that binds to a GFRAL polypeptide which has been chemically modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or a GFRAL antibody may be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide. A derivative of a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or a GFRAL antibody may be chemically modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Further, a derivative of a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or a GFRAL antibody may contain one or more non-classical amino acids. A polypeptide derivative possesses a similar or identical function as a GFRAL polypeptide, a fragment of a GFRAL polypeptide, or a GFRAL antibody described herein.

The term “involuntary body weight loss” refers to the unintended loss of body weight that is observed in many conditions such as cachexia, liver cirrhosis, hyperthyroidism, chronic kidney disease, Parkinson's disease, cancer, eating disorder, and sarcopenia.

The term “cachexia” refers to wasting syndrome that is marked with loss of weight, muscle atrophy, fatigue, weakness, and significant loss of appetite in someone who is not actively trying to lose weight. Cachexia can greatly contribute to morbidity of patients suffering from some chronic diseases (e.g., cancer, chronic renal disease, chronic inflammatory disease, muscle wasting, such as muscular dystrophy, and anorexia nervosa). For example, in late stage cancer, cachexia is common (occurring in most terminally ill cancer patients), and is responsible for about a quarter of all cancer-related deaths.

Compositions and Methods of Making the Same

Binding proteins such as antibodies that bind to GFRAL (e.g., human GFRAL) are provided. Antibodies of the present disclosure are useful, for example, for the diagnosis or treatment of GDF15-mediated diseases, disorders, or conditions. In certain embodiments, antibodies of the present disclosure are useful for the diagnosis or treatment of a disease, disorder, or condition, such as involuntary body weight loss, including, but not limited to, involuntary body weight loss in a subject suffering from cachexia or a chronic disease (e.g., liver cirrhosis, hyperthyroidism, Parkinson's disease, cancer, chronic renal disease, chronic obstructive pulmonary disease, AIDS, tuberculosis, chronic inflammatory disease, sepsis, muscle wasting, and anorexia nervosa) or broadly any disease, disorder, or condition in which it is desirable to inhibit the in vivo effects of GDF15.

Provided herein are antibodies (e.g., monoclonal antibodies) that bind to a GFRAL polypeptide, a GFRAL polypeptide fragment, GFRAL peptide, or a GFRAL epitope. In some embodiments, the anti-GFRAL antibodies bind to the extracellular domain (ECD) of GFRAL. Also provided are antibodies that competitively block an anti-GFRAL antibody provided herein from binding to a GFRAL polypeptide. The anti-GFRAL antibodies provided herein can also be conjugated or recombinantly fused to a diagnostic agent, detectable agent or therapeutic agent. Further provided are compositions comprising an anti-GFRAL antibody.

Also provided herein are isolated nucleic acid molecules encoding an immunoglobulin heavy chain, light chain, VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of anti-GFRAL antibodies that bind to a GFRAL polypeptide, a GFRAL polypeptide fragment, a GFRAL peptide or a GFRAL epitope. Further provided are vectors and host cells comprising nucleic acid molecules encoding anti-GFRAL antibodies that bind to a GFRAL polypeptide, a GFRAL polypeptide fragment, a GFRAL peptide or a GFRAL epitope. Also provided are methods of making antibodies that bind to a GFRAL polypeptide, a GFRAL polypeptide fragment, a GFRAL peptide or a GFRAL epitope.

Methods of using the anti-GFRAL antibodies are provided herein. The methods include treating, preventing or alleviating a GDF15-mediated disease, disorder or condition, including treating, preventing or alleviating one or more symptoms of a GDF15-mediated disease, disorder or condition in a subject (e.g., patient). Non limiting examples of GDF15-mediated diseases, disorders, or conditions include involuntary weight loss, a waisting disease, involuntary body weight loss in a subject suffering from cachexia or a chronic disease (e.g., liver cirrhosis, hyperthyroidism, Parkinson's disease, cancer, chronic renal disease, chronic obstructive pulmonary disease, AIDS, tuberculosis, chronic inflammatory disease, sepsis, muscle wasting, and anorexia nervosa). Other of diseases, disorders, or conditions in which a subject can suffer from involuntary weight loss include eating disorders, muscular dystrophy or multiple sclerosis.

Anti-GFRAL Antibodies

In some embodiments, the present disclosure provides anti-GFRAL antibodies that may find use herein as therapeutic agents. Exemplary antibodies include polyclonal, monoclonal, humanized, human, bispecific, and heteroconjugate antibodies, as well as variants thereof having improved affinity or other properties.

In some embodiments, provided herein are antibodies that bind to GFRAL, including a GFRAL polypeptide, a GFRAL polypeptide fragment, a GFRAL peptide or a GFRAL epitope. In some embodiments the anti-GFRAL antibodies are humanized antibodies (e.g., comprising human constant regions) that bind GFRAL, including GFRAL polypeptide, a GFRAL polypeptide fragment, a GFRAL peptide or a GFRAL epitope.

In some embodiments, an anti-GFRAL antibody comprises a VH region, VL region, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of any one of the monoclonal antibodies described herein (e.g., 1C1, 3P10, 12A3, 5F12, 5A20, 8D8, 17J16, 25M22, 2B8, 22N5, 2I23, 6N16, 1B3, 19K19, 2B3, 8C10, 2A9, 24G2, 6G9, 2B11, 1A3, P1B6, P1H8, or P8G4), such as an amino acid sequence depicted in Tables 1-24. Accordingly, in some embodiments, the isolated antibody or functional fragment thereof provided herein comprises one, two, and/or three heavy chain CDRs and/or one, two, and/or three light chain CDRs from: (a) the antibody designated 1C1; (b) the antibody designated 3P10; (c) the antibody designated 12A3; (d) the antibody designated 5F12; (e) the antibody designated 5A20; (f) the antibody designated 8D8; (g) the antibody designated 17J16; (h) t the antibody designated 25M22; (i) the antibody designated 2B8; (j) the antibody designated 22N5; (k) the antibody designated 2123; (l) the antibody designated 6N16; (m) the antibody designated 1B3; (n) the antibody designated 19K19; (o) the antibody designated 2B3; (p) the antibody designated 8C10; (q) the antibody designated 2A9; (r) the antibody designated 24G2; (s) the antibody designated 6G9; (t) the antibody designated 2B11; (u) the antibody designated 1A3; (v) the antibody designated P1B6; (w) the antibody designated P1H8; (x) the antibody designated P8G4; or the antibody designated 1C1 to P8G4, as shown in Tables 1-24.

The antibody designated 1C1 comprises a VH sequence that is SEQ ID NO: 1 and a VL sequence that is SEQ ID NO: 2.

The antibody designated 3P10 comprises a VH sequence that is SEQ ID NO: 3 and a VL sequence that is SEQ ID NO: 4,

The antibody designated 12A3 comprises a VH sequence that is SEQ ID NO: 5 and a VL sequence that is SEQ ID NO: 6.

The antibody designated 5F12 comprises a VH sequence that is SEQ ID NO: 7 and a VL sequence that is SEQ ID NO: 8.

The antibody designated 5A20 comprises a VH sequence that is SEQ ID NO: 9 and a VL sequence that is SEQ ID NO: 10.

The antibody designated 8D8 comprises a VH sequence that is SEQ ID NO: 11 and a VL sequence that is SEQ ID NO: 12.

The antibody designated 17J16 comprises a VH sequence that is SEQ ID NO: 13 and a VL sequence that is SEQ ID NO: 14.

The antibody designated 25M22 comprises a VH sequence that is SEQ ID NO: 15 and a VL sequence that is SEQ ID NO: 16.

The antibody designated 2B8 comprises a VH sequence that is SEQ ID NO: 17 and a VL sequence that is SEQ ID NO: 18.

The antibody designated 22N5 comprises a VH sequence that is SEQ ID NO: 19 and a VL sequence that is SEQ ID NO: 20.

The antibody designated 2123 comprises a VH sequence that is SEQ ID NO: 21 and a VL sequence that is SEQ ID NO: 22.

The antibody designated 6N16 comprises a VH sequence that is SEQ ID NO: 23 and a VL sequence that is SEQ ID NO: 24.

The antibody designated 1B3 comprises a VH sequence that is SEQ ID NO: 25 and a VL sequence that is SEQ ID NO: 26.

The antibody designated 19K19 comprises a VH sequence that is SEQ ID NO: 27 and a VL sequence that is SEQ ID NO: 28.

The antibody designated 2B3 comprises a VH sequence that is SEQ ID NO: 29 and a VL sequence that is SEQ ID NO: 30.

The antibody designated 8C10 comprises a VH sequence that is SEQ ID NO: 31 and a VL sequence that is SEQ ID NO: 32.

The antibody designated 2A9 comprises a VH sequence that is SEQ ID NO: 33 and a VL sequence that is SEQ ID NO: 34.

The antibody designated 24G2 comprises a VH sequence that is SEQ ID NO: 35 and a VL sequence that is SEQ ID NO: 36.

The antibody designated 6G9 comprises a VH sequence that is SEQ ID NO: 37 and a VL sequence that is SEQ ID NO: 38.

The antibody designated 2B11 comprises a VH sequence that is SEQ ID NO: 39 and a VL sequence that is SEQ ID NO: 40.

The antibody designated 1A3 comprises a VH sequence that is SEQ ID NO: 480 and a VL sequence that is SEQ ID NO: 481.

The antibody designated P1B6 comprises a VH sequence that is SEQ ID NO: 482 and a VL sequence that is SEQ ID NO: 483.

The antibody designated P1H8 comprises a VH sequence that is SEQ ID NO: 484 and a VL sequence that is SEQ ID NO: 485.

The antibody designated P8G4 comprises a VH sequence that is SEQ ID NO: 486 and a VL sequence that is SEQ ID NO: 487.

TABLE 1 Antibody 1C1 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GFSLNDYGVH GFSLNDYG DYGVH GFSLNDY NDYGVH GFSLNDYGVH Seq. (SEQ ID NO: 41) (SEQ ID NO: 42 (SEQ ID NO: 43) (SEQ ID (SEQ ID (SEQ ID NO: 44) NO: 45) NO: 41) VH CDR2 VIWSGGRTDYNAAFIS IWSGGRT VIWSGGRTDYNAAFIS SGG WLGVIWSGGRTD VIWSGGRTD (SEQ ID NO: 132) (SEQ ID NO: 133) (SEQ ID NO: 132) (SEQ ID (SEQ ID (SEQ ID NO: 134) NO: 135) NO: 136) VH CDR3 WALYFLYGGSMDY ARWALYFLYGGSMDY WALYFLYGGSMDY ALYFLYGG ARWALYFLYGGS WALYFLYGGS (SEQ ID NO: 221) (SEQ ID NO: 122) (SEQ ID NO: 221) SMD MD MDY (SEQ ID (SEQ ID (SEQ ID NO: 223) NO: 224) NO: 221) VL CDR VL CDR1 RSSQSLVHSSGITYLH QSLVHSSGITY RSSQSLVHSSGITYLH SQSLVHSS VHSSGITYLHWY RSSQSLVHSS Seq. (SEQ ID NO: 297) (SEQ ID NO: 298) (SEQ ID NO: 297) GITY (SEQ ID GITYLH (SEQ ID NO: 300) (SEQ ID NO: 299) NO: 297) VL CDR2 KLSNRFS KLS KLSNRFS KLS LLIYKLSNRF KLSNRFS (SEQ ID NO: 373) (SEQ ID NO: 374) (SEQ ID NO: 373) (SEQ ID (SEQ ID (SEQ ID NO: 374) NO: 375) NO: 373) VL CDR3 SQSTHVPPWT SQSTHVPPWT SQSTHVPPWT STHVPPW SQSTHVPPW SQSTHVPPWT (SEQ ID NO: 423) (SEQ ID NO: 423) (SEQ ID NO: 423) (SEQ ID (SEQ ID (SEQ ID NO: 424) NO: 425) NO: 423) VH Sequence: QMQLKQSGPGLVQPSQSLSITCTVSGFSLNDYGVHWIRQSPGKLGEWLGVIWSGGRTDYNAAFISRLSISKDNSKSQVFFKMSSLQPQDTAIYYCA RWALYFLYGGSMDYWGQGTSVTVSS (SEQ ID NO: 1) VL Sequence: DVVLTQTPLSLPVSPGDQASISCRSSQSLVHSSGITYLHWYLQKPGQSPKLLIYKLSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQS THVPPWTFGGGTKLEIK (SEQ ID NO: 2)

TABLE 2 Antibody 3P10 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTDYGVI GYTFTDYG GYGVI GYTFTDY TDYGVI GYTFTDYGVI Seq. (SEQ ID NO: 46) (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 47) NO: 48) NO: 49) NO: 50) NO: 46) VH CDR2 WINTYTGEPTYADDLKG INTYTGEP WINTYTGEPTYA TYTG WMGWINTYTGEPT WINTYTGEPT (SEQ ID NO: 137) (SEQ ID DDLKG (SEQ ID (SEQ ID (SEQ ID NO: 138) (SEQ ID NO: 139) NO: 140) NO: 141) NO: 137) VH CDR3 RYGPEDIDY ARRYGPEDIDY RYGPEDIDY YGPEDID ARRYGPEDID RYGPEDIDY (SEQ ID NO: 225) (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 226) NO: 225) NO: 27) NO: 228) NO: 225) VL CDR VL CDR1 RASESVDNYGISFMS ESVDNYGISF RASESVDNYGIS SESVDNYGISF DNYGISFMSWF RASESVDNYG Seq. (SEQ ID NO: 301) (SEQ ID FMS (SEQ ID (SEQ ID ISFMS NO: 302) (SEQ ID NO: 303) NO: 304) (SEQ ID NO: 301) NO: 301) VL CDR2 AASHQGS AAS AASHQGS AAS LLIYAASHQG AASHQGS (SEQ ID NO: 376) (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 377) NO: 376) NO: 377) NO: 378) NO: 376) VL CDR3 LQSKEVPWT LQSKEVPWT LQSKEVPWT SKEVPW LQSKEVPW LQSKEVPWT (SEQ ID NO: 426) (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 426) NO: 426) NO: 427) NO: 428) NO: 426) VH Sequence: QIQLVQSGPELKKPGETVKISCKASGYTFTDYGVIWVKQAPGKALKWMGWINTYTGEPTYADDLKGRFAFSLETSASSASLQINNLKNEDTAT YFCARRYGPEDIDYWGQGTTLVTVSS (SEQ ID NO: 3) VL Sequence: DIVLTQSPVSLAVSLGQRATISCRASESVDNYGISFMSWFQQKPGQPPKLLIYAASHQGSGVPARFSGSGSGTDFSLNIHPMEEDDSAMYFCL QSKEVPWTFGGGTKLEIK (SEQ ID NO: 4)

TABLE 3 Antibody 12A3 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYPFTIYGMN GYPFTIYG IYGMN GYPFTIY TIYGMN GYPFTIYGMN Seq. (SEQ ID NO: 51) (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 55) (SEQ ID NO: 51) NO: 52) NO: 53) NO: 54) VH CDR2 WINTYSGVPTYADDF INTYSGVP WINTYSGVPTY TYSG WMGWINTYSGVPT WINTYSGVPT KG (SEQ ID ADDFKG (SEQ ID (SEQ ID NO: 145) (SEQ ID NO: 146) (SEQ ID NO: NO: 143) (SEQ ID NO: 144) 142) NO: 142) VH CDR3 ATGNY ASATGNY ATGNY TGN ASATGN ATGNY (SEQ ID NO: 229) (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 232) (SEQ ID NO: 229) NO: 230) NO: 229) NO: 231) VL CDR VL CDR1 RASQDIGSSLN QDIGSS RASQDIGSSLN SQDIGSS GSSLNWL RASQDIGSSLN Seq. (SEQ ID NO: 305) (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 308) (SEQ ID NO: 305) NO: 306) NO: 305) NO: 307) VL CDR2 ATSSLDS ATS ATSSLDS ATS TLIYATSSLD ATSSLDS (SEQ ID NO: 379) (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 381) (SEQ ID NO: 379) NO: 380) NO: 379) NO: 380) VL CDR3 LQYASSPYT LQYASSPYT LQYASSPYT YASSPY LQYASSPY LQYASSPYT (SEQ ID NO: 429) (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 431) (SEQ ID NO: 429) NO: 429) NO: 429) NO: 430) VH Sequence: QIQLVQSGPELKKPGETVKISCKASGYPFTIYGMNWVEQAPGKGLKWMGWINTYSGVPTYADDFKGRFAFSLETSASTAYLQINNLKDEDTATY FCASATGNYWGQGTTLVTVSS (SEQ ID NO: 5) VL Sequence: DIQMTQSPSSLSASLGERVSLTCRASQDIGSSLNWLQQEPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSLESEDFVDYYCLQYASS PYTFGGGTKVEIK (SEQ ID NO: 6)

TABLE 4 Antibody 5F12 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTDYYIN GYTFTDYY DYYIN GYTFTDY TDYYIN GYTFTDYYIN Seq. (SEQ ID NO: 56) (SEQ ID NO: 57) (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 56) NO: 58) NO: 49) NO: 59) VH CDR2 RIYPGNGNTYHNEKFK IYPGNGNT RIYPGNGN PGNG WIARIYPG RIYPGNGNTY G (SEQ ID NO: 148) THHNEKFK (SEQ ID NGNTY (SEQ ID NO: 151) (SEQ ID NO: 147) G NO: 149) (SEQ ID (SEQ ID NO: 150) NO: 47) VH CDR3 EGLYYDYDRYFDY AREGLYYDYDRYFDY EGLYYDYD GLYYDYDR AREGLYYD EGLYYDYDRYFDY (SEQ ID NO: 233) (SEQ ID NO: 234) RYFDY YFD YDRYFED (SEQ ID NO: 233) (SEQ ID (SEQ ID (SEQ ID NO: 233) NO: 235) NO: 236) VL CDR VL CDR1 RASESVDTYGNSFMH ESVDTYGNSF RASESVDT SESVDTYG DTYGNSFM RASESVDTYGNSFMH Seq. (SEQ ID NO: 309) (SEQ ID NO: 310) YGNSFMH NSF HWY (SEQ ID NO: 309) (SEQ ID (SEQ ID (SEQ ID NO: 309) NO: 311) NO: 312) VL CDR2 LASNLES LAS LASNLES LAS LLIYLASN (SEQ ID NO: 382) (SEQ ID NO: 383) (SEQ ID (SEQ ID LE LASNLES NO: 382) NO: 383) (SEQ ID (SEQ ID NO: 382) NO: 384) VL CDR3 HQNNEDPPA HQNNEDPPA HQNNEDPP NNEDPP HQNNEDPP HQNNEDPPA (SEQ ID NO: 432) (SEQ ID NO: 432) A (SEQ ID (SEQ ID (SEQ ID NO: 432) (SEQ ID NO: 433) NO: 434) NO: 432) VH Sequence: QVQLKQSGTELVRPGASVKLSCKASGYTFTDYYINWVKQRPGQGLEWIARIYPGNGNTYHNEKFKGKATLTAEKSSSTAYMQLSSLTSEDS AVYFCAREGLYYDYDRYFDYWGQGTALTVSS (SEQ ID NO: 7) VL Sequence: NIVLTQSPASLAVSLGQRATISCRASESVDTYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTDFTLTIDPVEADDAATYY CHQNNEDPPAFGGGTKLEIK (SEQ ID NO: 8)

TABLE 5 Antibody 5A20 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTDYWIE GYTFTDYW DYWIE GYTFTDY TDYWIE GYTFTDYWIE Seq. (SEQ ID (SEQ ID NO: 61) (SEQ ID NO: 62) (SEQ ID (SEQ ID (SEQ ID NO: 60) NO: 49) NO: 63) NO: 60) VH CDR2 EILLGSDSIH ILLGSDSI EILLGSDSIHFNEKFK LGSD WIGEILLG EILLGSDSIH FNEKFKG (SEQ ID NO: 153) G (SEQ ID SDSIH (SEQ ID (SEQ ID (SEQ ID NO: 152) NO: 154) (SEQ ID NO: 156) NO: 152) NO: 155) VH CDR3 QDWNWYFDV VRQDWNWYFDV QDWNWYFDV DWNWYFD VRQDWNWY QDWNWYFDV (SEQ ID (SEQ ID NO: 238) (SEQ ID NO: 237) (SEQ ID FD (SEQ ID NO: 237) NO: 239) (SEQ ID NO: 237) NO: 240) VL CDR VL CDR1 KSSQSLLDFD QSLLDFDGKTY KSSQSLLDFDGKTYLN SQSLLDF LDFDGKTY KSSQSLLDFD Seq. GKTYLN (SEQ ID NO: 314) (SEQ ID NO: 313) DGKTY LNWY GKTYLN (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 313) NO: 315) NO: 316) NO: 313) VL CDR2 LVSKLDS LVS LVSKLDS LVS RLFYLVSK LVSKLDS (SEQ ID (SEQ ID NO: 386) (SEQ ID NO: 385) (SEQ ID LD (SEQ ID NO: 385) NO: 386) (SEQ ID NO: 385) NO: 387) VL CDR3 WQGTHFPRT WQGTHFPRT WQGTHFPRT GTHFPR WQGTHFPR WQGTHFPRT (SEQ ID (SEQ ID NO: 435) (SEQ ID NO: 435) (SEQ ID (SEQ ID (SEQ ID NO: 435) NO: 436) NO: 437) NO: 435) VH Sequence: QVQLQQSGPELMKPGASVILSCKAIGYTFTDYWIEWVKERPGHGLEWIGEILLGSDSIHFNEKFKGKATISADTSSNTAYMQLSSLT TEDSAIYYCVRQDWNWYFDVWGTGTTVTVSS (SEQ ID NO: 9) VL Sequence: DVVMTQTPLTLSVTIGHPASISCKSSQSLLDFDGKTYLNWLFQRPGQSPKRLFYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAED LGVYYCWQGTHFPRTFGGGTKLEIK (SEQ ID NO: 10)

TABLE 6 Antibody 8D8 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GFSLSRYSVH GFSLSRYS RYSVH GFSLSRY SRYSVH GFSLSRYSVH Seq. (SEQ ID NO: 64) (SEQ ID (SEQ ID NO: 66) (SEQ ID (SEQ ID (SEQ ID NO: 64) NO: 65) NO: 67) NO: 68) VH CDR2 MIWGFGSTDYNSALKS IWGFGST MIWGFGSTDYNSALKS GFG WLGMIWGF MIWGFGSTD (SEQ ID NO: 157) (SEQ ID (SEQ ID NO: 157) (SEQ ID GSTD (SEQ ID NO: 160) NO: 220) NO: 158) (SEQ ID NO: 159) VH CDR3 IHTTAGSY ARIHTTAG IHTTAGSY HTTAGA ARIHTTAG IHTTAGSY (SEQ ID NO: 241) SY (SEQ ID NO: 241) (SEQ ID S (SEQ ID NO: 241) (SEQ ID NO: 243) (SEQ ID NO: 242) NO: 244) VL CDR VL CDR1 KASQNVGTNVA QNVGTN KASQNVGTNVA SQNVGTN GTNVAWY KASQNVGTNVA Seq. (SEQ ID NO: 317) (SEQ ID (SEQ ID NO: 317) (SEQ ID (SEQ ID (SEQ ID NO: 317) NO: 318) NO: 319) NO: 320) VL CDR2 STSYRYS STS STSYRYS STS ALVYSTSY STSYRYS (SEQ ID NO: 388) (SEQ ID (SEQ ID NO: 388) (SEQ ID RY (SEQ ID NO: 388) NO: 389) NO: 389) (SEQ ID NO: 390) VL CDR3 HQYNSYPLT HQYNSYPL HQYNSYPLT YNSYPL HQYNSYPL HQYNSYPLT (SEQ ID NO: 438) T (SEQ ID NO: 438) (SEQ ID (SEQ ID (SEQ ID NO: 438) (SEQ ID NO: 439) NO: 440) NO: 438) VH Sequence: QVQLKESGPGLVAPSQSLSITCTVSGFSLSRYSVHWVRQPPGKGLEWLGMIWGFGSTDYNSALKSRLSITKDNSKSQFFLKMNSLQTDDTA MYYCARIHTTAGSYWGQGTLVTVSA (SEQ ID NO: 11) VL Sequence: DIVMTQSQKFMSTSIGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALVYSTSYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCHQY NSYPLTFGAGTKLELK (SEQ ID NO: 12)

TABLE 7 Antibody 17J16 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTDYWIH GYTFTDWY DYWIH GYTFTDY TDYWIH GYTFTDYW Seq. (SEQ ID NO: 69) (SEQ ID NO: 61) (SEQ ID (SEQ ID NO: 49) (SEQ ID IH NO: 70) NO: 71) (SEQ ID NO: 69) VH CDR2 YINPNSNYAEYNQKFK INPNSNYA YINPNSNY PNSN WIGYINP YINPNSNY V (SEQ ID NO: 162) AEYNQKFK (SEQ ID NO: 163) NSNYAE AE (SEQ ID NO: 161) V (SEQ ID (SEQ ID (SEQ ID NO: 164) NO: 165) NO: 161) VH CDR3 FDWNWYFHV ARFDWNWYFHV FDWNWYFH DWNWYFH ARFDWNWY FDWNWYFH (SEQ ID NO: 246) (SEQ ID NO: 246) V (SEQ ID NO: 247) FH V (SEQ ID (SEQ ID (SEQ ID NO: 245) NO: 248) NO: 245) VL CDR VL CDR1 KSSQSLSDSDGKTYLN QSLSDSDGKTY KSSQSLSD SQSLSDSDGKTY SDSDGKTY KSSQSLSD Seq. (SEQ ID NO: 321) (SEQ ID NO: 322) SDGKTYLN (SEQ ID NO: 323) LNWL SDGKTYLN (SEQ ID (SEQ ID (SEQ ID NO: 321) NO: 324) NO: 321) VL CDR2 LVSRLGS LVS LVSRLGS LVS RLIYLVSR (SEQ ID NO: 391) (SEQ ID NO: 386) (SEQ ID (SEQ ID NO: 386) LG LVSRLGS NO: 391) (SEQ ID (SEQ ID NO: 392) NO: 391) VL CDR3 WQGTHFPQT WQGTHFPQT WQGTHFPQ GTHFPQ WQGTHFPQ WQGTHFPQ (SEQ ID NO: 441) (SEQ ID NO: 441) T (SEQ ID NO: 442) (SEQ ID T (SEQ ID NO: 443) (SEQ ID NO: 441) NO: 441) VH Sequence: QVQLQQSGAELAKPGASVKMSCKTSGYTFTDYWIHWVKQRPGQGLEWIGYINPNSNYAEYNQKFKVKATLTADKSSSTAYLQLSRLTSEDS AVYYCARFDWNWYFHVWGAGSTVTVSS (SEQ ID NO: 13) VL Sequence: DVALTQIPLTLSVTVGQPASISCKSSQSLSDSDGKTYLNWLLQKPGQSPKRLIYLVSRLGSGVPDRFTGSGSGADFTLKISRVEAEDLGVY YCWQGTHFPQTFGGGTKLEIK (SEQ ID NO: 14)

TABLE 8 Antibody 25M22 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTSYWV GYTFTSWY SYWVN GYTFTSY TSYWVN GYTFTSYW Seq. N (SEQ ID NO: 73) (SEQ ID NO: 74) (SEQ ID (SEQ ID NO: 76) VN (SEQ ID NO: 75) (SEQ ID NO: 72) NO: 72) VH CDR2 RIYPGDGDT IYPGDGDT RIYPGDGDTNYNGKFK PGDG WIGRIYPGDGDTN RIYPGDGD NYNGKFKG (SEQ ID NO: 167) G (SEQ ID (SEQ ID NO: 169) TN (SEQ ID (SEQ ID NO: 166) NO: 168) (SEQ ID NO: 166) NO: 170) VH CDR3 AYLLRLRRT ARAYLLRLRRTGYYAM AYLLRLRRTGYYAMDY YLLRLRRT ARAYLLRLRRTGYYAM AYLLRLRR GYYAMDY DY (SEQ ID NO: 249) GYYAMD (SEQ ID NO: 252) TGYYAMDY (SEQ ID (SEQ ID NO: 250) (SEQ ID (SEQ ID NO: 249) NO: 251) NO:249) VL CDR VL CDR1 KSTKSLLNS KSLLNSDEFTY KSTKSLLNSDEFTYLD TKSLLNSD LNSDEFTYLDWY KSTKSLLN Seq. DEFTYLD (SEQ ID NO: 326) (SEQ ID NO: 325) EFTY (SEQ ID NO: 328) SDEFTYLD (SEQ ID (SEQ ID (SEQ ID NO: 325) NO: 327) NO: 325) VL CDR2 LVSNRFS LVS LVSNRFS LVS (SEQ ID (SEQ ID NO: 386) (SEQ ID NO: 393) (SEQ ID LLIFLVSNRF LVSNRFS NO: 393) NO: 386) (SEQ ID NO: 394) (SEQ ID NO: 393) VL CDR3 FQSNYLPYT FQSNYLPYT FQSNYLPYT SNYLPY FQSNYLPY FQSNYLPY (SEQ ID (SEQ ID NO: 444) (SEQ ID NO: 444) (SEQ ID (SEQ ID NO: 446) T NO: 444) NO: 445) (SEQ ID NO: 444) VH Sequence: QVQLQQSGPDLVKPGASVKISCKASGYTFTSYWVNWMKQRPGKGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSA VYFCARAYLLRLRRTGYYAMDYWGQGTSVTVSS (SEQ ID NO: 15) VL sequence: DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDEFTYLDWYLQKPGQSPQLLIFLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYY CFQSNYLPYTFGGGTKLEIK (SEQ ID NO: 16)

TABLE 9 Antibody 2B8 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTTYGM GYTFTTYG TYGMS GYTFTTY TTYGMS GYTFTTYGMS Seq. S (SEQ ID NO: 78) (SEQ ID (SEQ ID (SEQ ID NO: 81) (SEQ ID NO: 77) (SEQ ID NO: 79) NO: 80) NO: 77) VH CDR2 WINTYSGVP INTYSGVP WINTYSGV TYSG WMGWINTYSGVPT WINTYSGVPT TFVDDFRG (SEQ ID NO: 143) PTFVDDFR (SEQ ID (SEQ ID NO: 145) (SEQ ID NO: 146) (SEQ ID G NO: 144) NO: 171) (SEQ ID NO: 171) VH CDR3 RSSYYPYWY ARRSSYYPYWYFDV RSSYYPYW SSYYPYWY ARRSSYYPYWYFD RSSYYPYWYFDV FDV (SEQ ID NO: 254) YFDV FD (SEQ ID NO: 256) (SEQ ID NO: 253) (SEQ ID (SEQ ID (SEQ ID NO: 253) NO: 253) NO: 255) VL CDR VL CDR1 RPSENIYSY ENIYSY RPSENIYS SENIYSY YSYLTWF RPSENIYSYLT Seq. LT (SEQ ID NO: 330) YLT (SEQ ID (SEQ ID NO: 332) (SEQ ID NO: 329) (SEQ ID (SEQ ID NO: 331) NO: 329) NO: 329) VL CDR2 NAQTLAE NAQ NAQTLAE NAQ LLVYNAQTLA NAQTLAE (SEQ ID (SEQ ID NO: 396) (SEQ ID (SEQ ID (SEQ ID NO: 397) (SEQ ID NO: 395) NO: 395) NO: 395) NO: 396) VL CDR3 QHYYGYPFT QHYYGYPFT QHYYGYPF YYGYPF QHYYGYPF QHYYGYPFT (SEQ ID (SEQ ID NO: 447) T (SEQ ID (SEQ ID NO: 449) (SEQ ID NO: 447) NO: 447) (SEQ ID NO: 448) NO: 447) VH Sequence: QIQLVQSGPELKKPGETVKISCKASGYTFTTYGMSWVKQAPGKIFKWMGWINTYSGVPTFVDDFRGRFAFSLETSASTAYLQIGNLKNEDTA TYFCARRSSYYPYWYFDVWGTGTTVTVSS (SEQ ID NO: 17) VL Sequence: DIQMTQSPASLSASVGETVTITCRPSENIYSYLTWFQQEQGKSPQLLVYNAQTLAEGVPSRFSGSGSGTHFSLKINSLQPEDFGTYYCQHYY GYPFTFGSGTKLEIK (SEQ ID NO: 18)

TABLE 10 Antibody 22N5 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTDYSMH GYTFTDYS DYSMH GYTFTDY TDYSMH GYTFTDYS Seq. (SEQ ID NO: 82) (SEQ ID NO: 83) (SEQ ID NO: 84) (SEQ ID (SEQ ID MH NO: 49) NO: 85) (SEQ ID NO: 82) VH CDR2 WINTETGEPTYADDFK INTETGEP WINTETGEPTYADDFK TETG WMGWINTE WINTETGE G (SEQ ID NO: 173) G (SEQ ID TGEPT PT (SEQ ID NO: 172) (SEQ ID NO: 172) NO: 174) (SEQ ID (SEQ ID NO: 175) NO: 176) VH CDR3 GTLNY VKGTLNY GTLNY TLN VKGTLN GTLNY (SEQ ID NO: 257) (SEQ ID NO: 258) (SEQ ID NO: 257) (SEQ ID (SEQ ID (SEQ ID NO: 259) NO: 260) NO: 257) VL CDR VL CDR1 KASQDIKSYLN QDIKSY KASQDIKSYLN SQDIKSY KSYLNWF KASQDIKS Seq. (SEQ ID NO: 333) (SEQ ID NO: 334) (SEQ ID NO: 333) (SEQ ID (SEQ ID YLN NO: 335) NO: 336) (SEQ ID NO: 333) VL CDR2 RTKRLVD RTK RTKRLVD RTK TLIYRTKR RTKRLVD (SEQ ID NO: 398) (SEQ ID NO: 399) (SEQ ID NO: 398) (SEQ ID LV (SEQ ID NO: 399) (SEQ ID NO: 398) NO: 400) VL CDR3 LQYVEFPLT LQYVEFPLT LQYVEFPLT YVEFPL LQYVEFPL LQYVEFPL (SEQ ID NO: 450) (SEQ ID NO: 450) (SEQ ID NO: 450) (SEQ ID (SEQ ID T NO: 451) NO: 452) (SEQ ID NO: 450) VH Sequence: QNQLVQSGPELKKPGEIVKISCKTSGYTFTDYSMHWVKKTPGKGFKWMGWINTETGEPTYADDFKGRFAFSLETSANTAHLQITNLKNEDT ATYFCVKGTLNYWGQGTTLVTVSS (SEQ ID NO: 19) VL Sequence: DIKMTQSPSSMYASLGERVTITCKASQDIKSYLNWFQQKPGKSPKTLIYRTKRLVDGVPSRFSGSGSGQDYSLTVSSLEYDDVGIYYCLQY VEFPLTFGDGTKLELK (SEQ ID NO: 20)

TABLE 11 Antibody 2I23 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYSFTSYNID GYSFTSYN SYNID GYSFTSY TSYNID GYSFTSYNID Seq. (SEQ ID NO: 86) (SEQ ID NO: 87) (SEQ ID NO: 88) (SEQ ID NO: 89) (SEQ ID NO: 90) (SEQ ID NO: 86) VH CDR2 WIFPGDGST IFPGDGST WIFPGDGSTKYNE PGDG WIGWIFPGDGSTK WIFPGDGSTK (SEQ ID NO: 177) (SEQ ID NO: 178) KFKG (SEQ ID NO: 168) (SEQ ID NO: 180) (SEQ ID NO: 181) (SEQ ID NO: 179) VH CDR3 SGIYYGSHFVY ARSGIYYGSHFVY SGIYYGSHFVY GIYYGSHFV ARSGIYYGSHFV SGIYYGSHFVY (SEQ ID NO: 261) (SEQ ID NO: 262) (SEQ ID NO: 261) (SEQ ID NO: 263) (SEQ ID NO: 264) (SEQ ID NO: 261) VL CDR VL CDR1 RSSQSLLDSDGKT QSLLDSDGKTY RSSQSLLDSDGKT SQSLLDSDGKTY LDSDGKTYLNWL RSSQSLLDSDGKT Seq. YLN (SEQ ID NO: 338) YLN (SEQ ID NO: 339) (SEQ ID NO: 340) YLN (SEQ ID NO: 337) (SEQ ID NO: 337) (SEQ ID NO: 337) VL CDR2 LVSKVDS LVS LVSKVDS LVS RLIYLVSKVD LVSKVDS (SEQ ID NO: 401) (SEQ ID NO: 386) (SEQ ID NO: 401) (SEQ ID NO: 386) (SEQ ID NO: 402) (SEQ ID NO: 401) VL CDR3 WQGTHFPLT WQGTHFPLT WQGTHFPLT GTHFPL WQGTHFPL WQGTHFPLT (SEQ ID NO: 453) (SEQ ID NO: 453) (SEQ ID NO: 453) (SEQ ID NO: 454) (SEQ ID NO: 455) (SEQ ID NO: 453) VH Sequence: QAQLQQSGAELVKPGASVKLSCKASGYSFTSYNIDWVRQRPEQGLEWIGWIFPGDGSTKYNEKFKGQATLTTDKSSSTTYIHLSRLTSEDSAVYF CARSGIYYGSHFVYWGQGTLVTVSA (SEQ ID NO: 21) VL Sequence: DVVMTQTPLTLSVTIGQSASISCRSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYFCWQ GTHFPLTFGAGTKLELK (SEQ ID NO: 22)

TABLE 12 Antibody 6N16 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTSYNIN GYTFTSYN SYNIN GYTFTSY TSYNIN GYTFTSYNIN Seq. (SEQ ID NO: 91) (SEQ ID NO: 92) (SEQ ID NO: 93) (SEQ ID NO: 75) (SEQ ID NO: 94) (SEQ ID NO: 91) VH CDR2 WIFPGDDSIKYNE IFPGDDSI WIFPGDDSIKYNE PGDD WIGWIFPGDDSIK WIFPGDDSIK NFRG (SEQ ID NO: 183) NFRG (SEQ ID NO: 184) (SEQ ID NO: 185) (SEQ ID NO: 186) (SEQ ID NO: 182) (SEQ ID NO: 182) VH CDR3 SGIFYGNNFAY ARSGIFYGNNFAY SGIFYGNNFAY GIFYGNNFA ARSGIFYGNNFA SGIFYGNNFAY (SEQ ID NO: 265) (SEQ ID NO: 266) (SEQ ID NO: 265) (SEQ ID NO: 267) (SEQ ID NO: 268) (SEQ ID NO: 265) VL CDR VL CDR1 KSSQSLLDGDGET QSLLDGDGETY KSSQSLLDGDGET SQSLLDGDGETY LDGDGETYLSWL KSSQSLLDGDGET Seq. YLS (SEQ ID NO: 342) YLS (SEQ ID NO: 343) (SEQ ID NO: 344) YLS (SEQ ID NO: 341) (SEQ ID NO: 341) (SEQ ID NO: 341) VL CDR2 LVSKLDS LVS LVSKLDS LVS RLIYLVSKLD LVSKLDS (SEQ ID NO: 385) (SEQ ID NO: 386) (SEQ ID NO: 385) (SEQ ID NO: 386) (SEQ ID NO: 403) (SEQ ID NO: 385) VL CDR3 CQSTHFPLT CQSTHFPLT CQSTHFPLT STHFPL CQSTHFPL CQSTHFPLT (SEQ ID NO: 456) (SEQ ID NO: 456) (SEQ ID NO: 456) (SEQ ID NO: 457) (SEQ ID NO: 458) (SEQ ID NO: 456) VH Sequence: QVQLQQSGSELVKPGTSMKLSCKASGYTFTSYNINWVRLRPEQGLEWIGWIFPGDDSIKYNENFRGKATLTTDKSSSTAYMHLSRLTSDDSAVYF CARSGIFYGNNFAYWGQGTLVTVSA (SEQ ID NO: 23) VL Sequence: DVVMTQAPLILSVTIGQPASISCKSSQSLLDGDGETYLSWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCCQST HFPLTFGAGTKLELK (SEQ ID NO: 24)

TABLE 13 Antibody 1B3 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GFTFTGYNIN GFTFTGYN GYNIN GFTFTGY TGYNIN GFTFTGYNIN Seq. (SEQ ID NO: 95) (SEQ ID NO: 96) (SEQ ID NO: 97) (SEQ ID NO: 98) (SEQ ID NO: 99) (SEQ ID NO: 95) VH CDR2 WIFPGDDNAKYNE IFPGDDNA WIFPGDDNAKYNE PGDD WIGWIFPGDDNAK WIFPGDDNAK KFKG (SEQ ID NO: 188) KFKG (SEQ ID NO: 184) (SEQ ID NO: 189) (SEQ ID NO: 190) (SEQ ID NO: 187) (SEQ ID NO: 187) VH CDR3 TPVLSNYFDY ARTPVLSNYFDY TPVLSNYFDY PVLSNYFD ARTPVLSNYFD TPVLSNYFDY (SEQ ID NO: 269) (SEQ ID NO: 270) (SEQ ID NO: 269) (SEQ ID NO: 271) (SEQ ID NO: 272) (SEQ ID NO: 269) VL CDR VL CDR1 KASQDISKYIS QDISKY KASQDISKYIS SQDISKY SKYISWY KASQDISKYIS Seq. (SEQ ID NO: 345) (SEQ ID NO: 346) (SEQ ID NO: 345) (SEQ ID NO: 347) (SEQ ID NO: 348) (SEQ ID NO: 345) VL CDR2 YTSTLQP YTS YTSTLQP YTS LLIHYTSTLQ YTSTLQP (SEQ ID NO: 404) (SEQ ID NO: 405) (SEQ ID NO: 404) (SEQ ID NO: 405) (SEQ ID NO: 406) (SEQ ID NO: 404) VL CDR3 LQYDNLYT LQYDNLYT LQYDNLYT YDNLY LQYDNLY LQYDNLYT (SEQ ID NO: 459) (SEQ ID NO: 459) (SEQ ID NO: 459) (SEQ ID NO: 460) (SEQ ID NO: 461) (SEQ ID NO: 459) VH Sequence: QVHLQQPGAELVKPGASVKLSCKASGFTFTGYNINWVRLRPEQGLEWIGWIFPGDDNAKYNEKFKGKATLTTDKSSNTAYMQLSRLTSEDSAVY FCARTPVLSNYFDYWGQGTTLTVSS (SEQ ID NO: 25) VL Sequence: DIQMTQSPSSLSASLGGKVTITCKASQDISKYISWYQHKPGKSPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLYTFG GGTKLEIK (SEQ ID NO: 26)

TABLE 14 Antibody 19K19 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYAFTSYWMN GYAFTSYW SYWMN GYAFTSY TSYWMN GYAFTSYWMN Seq. (SEQ ID NO: 100) (SEQ ID NO: 101) (SEQ ID NO: 102) (SEQ ID NO: 103) (SEQ ID NO: 104) (SEQ ID NO: 100) VH CDR2 RIYPGDGDTNYNG IYPGDGDT RIYPGDGDTNYNG PGDG WIGRIYPGDGDTN RIYPGDGDTN KFKG (SEQ ID NO: 167) KFKG (SEQ ID NO: 168) (SEQ ID NO: 169) (SEQ ID NO: 170) (SEQ ID NO: 166) (SEQ ID NO: 166) VH CDR3 AYLLRLRRTGYYA ARAYLLRLRRTGY AYLLRLRRTGYYA YLLRLRRTGYYAM ARAYLLRLRRTGY AYLLRLRRTGYYA MDY YAMDY MDY D YAMD MDY (SEQ ID NO: 249) (SEQ ID NO: 250) (SEQ ID NO: 249) (SEQ ID NO: 251) (SEQ ID NO: 252) (SEQ ID NO: 249) VL CDR VL CDR1 KSTKSLLNSDEFT KSLLNSDEFTY KSTKSLLNSDEFT TKSLLNSDEFTY LNSDEFTYLDWY KSTKSLLNSDEFT Seq. YLD (SEQ ID NO: 326) YLD (SEQ ID NO: 327) (SEQ ID NO: 328) YLD (SEQ ID NO: 325) (SEQ ID NO: 325) (SEQ ID NO: 325) VL CDR2 LVSNRFS LVS LVSNRFS LVS LLIYLVSNRF LVSNRFS (SEQ ID NO: 393) (SEQ ID NO: 386) (SEQ ID NO: 393) (SEQ ID NO: 386) (SEQ ID NO: 407) (SEQ ID NO: 393) VL CDR3 FQSNYLPYT FQSNYLPYT FQSNYLPYT SNYLPY FQSNYLPY FQSNYLPYT (SEQ ID NO: 444) (SEQ ID NO: 444) (SEQ ID NO: 444) (SEQ ID NO: 445) (SEQ ID NO: 446) (SEQ ID NO: 444) VH Sequence: QVQLQQSGPDLVKPGASVKISCKASGYAFTSYWMNWVKQRPGKGLEWIGRIYPGDGDTNYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAV YFCARAYLLRLRRTGYYAMDYWGQGTSVTVSS (SEQ ID NO: 27) VL Sequence: DVVLTQTPLSLPVNIGDQASISCKSTKSLLNSDEFTYLDWYLQKPGQSPQLLIYLVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQSN YLPYTFGGGTKLEIK (SEQ ID NO: 28)

TABLE 15 Antibody 2B3 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GFTFSDYFMF GFTFSDYF DYFMF GFTFSDY SDYFMF GFTFSDYFMF Seq. (SEQ ID NO: 105) (SEQ ID NO: 106) (SEQ ID NO: 107) (SEQ ID NO: 108) (SEQ ID NO: 109) (SEQ ID NO: 105) VH CDR2 YISNDGDSTYYPD ISNDGDST YISNDGDSTYYPD NDGD WVAYISNDGDST YISNDGDSTY TVQG (SEQ ID NO: 192) TVQG (SEQ ID NO: 193) Y (SEQ ID NO: 195) (SEQ ID NO: 191) (SEQ ID NO: 191) (SEQ ID NO: 194) VH CDR3 QGAQATLDY TRQGAQATLDY QGAQATLDY GAQATLD TRQGAQATLD QGAQATLDY (SEQ ID NO: 273) (SEQ ID NO: 274) (SEQ ID NO: 273) (SEQ ID NO: 275) (SEQ ID NO: 276) (SEQ ID NO: 273) VL CDR VL CDR1 SASSSVFYMH SSVFY SASSSVFYMH SSSVFY FYMHWY SASSSVFYMH Seq. (SEQ ID NO: 349) (SEQ ID NO: 350) (SEQ ID NO: 349) (SEQ ID NO: 351) (SEQ ID NO: 352) (SEQ ID NO: 349) VL CDR2 STSNLAS STS STSNLAS STS LLIYSTSNLA STSNLAS (SEQ ID NO: 408) (SEQ ID NO: 389) (SEQ ID NO: 408) (SEQ ID NO: 389) (SEQ ID NO: 409) (SEQ ID NO: 408) VL CDR3 HQWSST HQWSST HQWSST WSS HQWSS HQWSST (SEQ ID NO: 462) (SEQ ID NO: 462) (SEQ ID NO: 462) (SEQ ID NO: 463) (SEQ ID NO: 464) (SEQ ID NO: 462) VH Sequence: EVKLVESGGGLVQPGGSLKLSCAASGFTFSDYFMFWVRQTPEKRLEWVAYISNDGDSTYYPDTVQGRFTISRDNAKNTLYLQMSRLRSEDTAMY YCTRQGAQATLDYWGQGTTLTVSS (SEQ ID NO: 29) VL Sequence: QIVLTQSPAIMSASLGEEITLTCSASSSVFYMHWYQQKSGTSPKWYSTSNLASGIPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSTFGGG TKLEIK (SEQ ID NO: 30)

TABLE 16 Antibody 8C10 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFANYGLT GYTFANYG NYGLT GYTFANY ANYGLT GYTFANYGLT Seq. (SEQ ID NO: 110) (SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 113) (SEQ ID NO: 114) (SEQ ID NO: 110) VH CDR2 EIYPGSGHTHYNE IYPGSGHT EIYPGSGHTHYNE PGSG WIGEIYPGSGHTH EIYPGSGHTH DFKG (SEQ ID NO: 197) DFKG (SEQ ID NO: 198) (SEQ ID NO: 199) (SEQ ID NO: 200) (SEQ ID NO: 196) (SEQ ID NO: 196) VH CDR3 RIQLLLPVGGFVY ARRIQLLLPVGGF RIQLLLPVGGFVY IQLLLPVGGFV ARRIQLLLPVGGF RIQLLLPVGGFVY (SEQ ID NO: 277) VY (SEQ ID NO: 277) (SEQ ID NO: 279) V (SEQ ID NO: 277) (SEQ ID NO: 278) (SEQ ID NO: 280) VL CDR VL CDR1 RASQSISNNLH QSISNN RASQSISNNLH SQSISNN SNNLHWY RASQSISNNLH Seq. (SEQ ID NO: 353) (SEQ ID NO: 354) (SEQ ID NO: 353) (SEQ ID NO: 355) (SEQ ID NO: 356) (SEQ ID NO: 353) VL CDR2 YASQSIS YAS YASQSIS YAS LLIKYASQSI YASQSIS (SEQ ID NO: 410) (SEQ ID NO: 411) (SEQ ID NO: 410) (SEQ ID NO: 411) (SEQ ID NO: 412) (SEQ ID NO: 410) VL CDR3 QQSNSWPHT QQSNSWPHT QQSNSWPHT SNSWPH QQSNSWPH QQSNSWPHT (SEQ ID NO: 465) (SEQ ID NO: 465) (SEQ ID NO: 465) (SEQ ID NO: 466) (SEQ ID NO: 467) (SEQ ID NO: 465) VH Sequence: QVQLQQSGVELARPGAAVKLSCKASGYTFANYGLTWVKQRTGQGLEWIGEIYPGSGHTHYNEDFKGKATLTADRSSSTAYMELRSLTSEDSAVY FCARRIQLLLPVGGFVYWGQGTLVTVSA (SEQ ID NO: 31) VL Sequence: DFVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGVYFCQQSNSWPHT FGGGTKLEIK (SEQ ID NO: 32)

TABLE 17 Antibody 2A9 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GFTFSTYAMS GFTFSTYA TYAMS GFTFSTY STYAMS GFTFSTYAMS Seq. (SEQ ID NO: 115) (SEQ ID NO: 116) (SEQ ID NO: 117) (SEQ ID NO: 118) (SEQ ID NO: 119) (SEQ ID NO: 115) VH CDR2 SITSGGTTYYTDS ITSGGTT SITSGGTTYYTDS SGG WVASITSGGTTY SITSGGTTY VKG (SEQ ID NO: 202) VKG (SEQ ID NO: 134) (SEQ ID NO: 203) (SEQ ID NO: 204) (SEQ ID NO: 201) (SEQ ID NO: 201) VH CDR3 DGNFYYYGMDY ARDGNFYYYGMD DGNFYYYGMDY GNFYYYGMD ARDGNFYYYGMD DGNFYYYGMDY (SEQ ID NO: 281) Y (SEQ ID NO: 281) (SEQ ID NO: 283) (SEQ ID NO: 284) (SEQ ID NO: 281) (SEQ ID NO: 282) VL CDR VL CDR1 KASQNVGTAVA QNVGTA KASQNVGTAVA SQNVGTA GTAVAWY KASQNVGTAVA Seq. (SEQ ID NO: 357) (SEQ ID NO: 358) (SEQ ID NO: 357) (SEQ ID NO: 359) (SEQ ID NO: 360) (SEQ ID NO: 357) VL CDR2 SASNRFT SAS SASNRFT SAS ILIYSASNRF SASNRFT (SEQ ID NO: 413) (SEQ ID NO: 414) (SEQ ID NO: 413) (SEQ ID NO: 414) (SEQ ID NO: 415) (SEQ ID NO: 413) VL CDR3 QQYSSYFT QQYSSYFT QQYSSYFT YSSYF QQYSSYF QQYSSYFT (SEQ ID NO: 468) (SEQ ID NO: 468) (SEQ ID NO: 468) (SEQ ID NO: 469) (SEQ ID NO: 470) (SEQ ID NO: 468) VH Sequence: EVKLVESGGGLVKPGGSLKLSCAASGFTFSTYAMSWVRQTPEKRLEWVASITSGGTTYYTDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYC ARDGNFYYYGMDYWGQGTSVTVSS (SEQ ID NO: 33) VL Sequence: DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVAWYQQKPGQSPKILIYSASNRFTGVPDRFTGSGSGTDFTLTISNMQSEDLADYFCQQYSSYFT FGGGTKLELK (SEQ ID NO: 34)

TABLE 18 Antibody 24G2 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTTYWMH GYTFTTYW TYWMH GYTFTTY TTYWMH GYTFTTYWMH Seq. (SEQ ID NO: 120) (SEQ ID NO: 121) (SEQ ID NO: 122) (SEQ ID NO: 80) (SEQ ID NO: 123) (SEQ ID NO: 120) VH CDR2 MIHPNSGSSNYNE IHPNSGSS MIHPNSGSSNYNE PNSG WIGMIHPNSGSSN MIHPNSGSSN KFKN (SEQ ID NO: 206) KFKN (SEQ ID NO: 207) (SEQ ID NO: 208) (SEQ ID NO: 209) (SEQ ID NO: 205) (SEQ ID NO: 205) VH CDR3 SDYGFIPYFDY ARSDYGFIPYFDY SDYGFIPYFDY DYGFIPYFD ARSDYGFIPYFD SDYGFIPYFDY (SEQ ID NO: 285) (SEQ ID NO: 286) (SEQ ID NO: 285) (SEQ ID NO: 287) (SEQ ID NO: 288) (SEQ ID NO: 285) VL CDR VL CDR1 RASQSIGTSIH QSIGTS RASQSIGTSIH SQSIGTS GTSIHWY RASQSIGTSIH Seq. (SEQ ID NO: 361) (SEQ ID NO: 362) (SEQ ID NO: 361) (SEQ ID NO: 363) (SEQ ID NO: 364) (SEQ ID NO: 361) VL CDR2 YASESIS YAS YASESIS YAS LLIKYASESI YASESIS (SEQ ID NO: 416) (SEQ ID NO: 411) (SEQ ID NO: 416) (SEQ ID NO: 411) (SEQ ID NO: 417) (SEQ ID NO: 416) VL CDR3 QQSNSWPTFT QQSNSWPTFT QQSNSWPTFT SNSWPTF QQSNSWPTF QQSNSWPTFT (SEQ ID NO: 471) (SEQ ID NO: 471) (SEQ ID NO: 471) (SEQ ID NO: 472) (SEQ ID NO: 473) (SEQ ID NO: 471) VH Sequence: QVQLQQSGAELLKPGASVKLSCKASGYTFTTYWMHWVKQRPGQGLEWIGMIHPNSGSSNYNEKFKNKATLTVDKSSSTAYMQLSSLTSEDSAVY FCARSDYGFIPYFDYWGQGTTLTVS (SEQ ID NO: 35) VL Sequence: DILLTQSPAILSVSPGERVSFSCRASQSIGTSIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLIINSVESEDIADYYCQQSNSWPTFTF GAGTKLELK (SEQ ID NO: 36)

TABLE 19 Antibody 6G9 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTSYWMQ GYTFTSYW SYWMQ GYTFTSY TSYWMQ GYTFTSYWMQ Seq. (SEQ ID NO: 124) (SEQ ID NO: 73) (SEQ ID NO: 125) (SEQ ID NO: 75) (SEQ ID NO: 126) (SEQ ID NO: 124) VH CDR2 EIDPSDSYTNYNQ IDPSDSYT EIDPSDSYTNYNQ PSDS WIGEIDPSDSYTN EIDPSDSYTN KFKG (SEQ ID NO: 211) KFKG (SEQ ID NO: 212) (SEQ ID NO: 213) (SEQ ID NO: 214) (SEQ ID NO: 210) (SEQ ID NO: 210) VH CDR3 PLDRSAYYFDY ARPLDRSAYYFDY PLDRSAYYFDY LDRSAYYFD ARPLDRSAYYFD PLDRSAYYFDY (SEQ ID NO: 289) (SEQ ID NO: 290) (SEQ ID NO: 289) (SEQ ID NO: 291) (SEQ ID NO: 292) (SEQ ID NO: 289) VL CDR VL CDR1 RASESVDFSGNSF ESVDFSGNSF RASESVDFSGNSF SESVDFSGNSF DFSGNSFMHWY RASESVDFSGNSF Seq. MH (SEQ ID NO: 366) MH (SEQ ID NO: 367) (SEQ ID NO: 368) MH (SEQ ID NO: 365) (SEQ ID NO: 365) (SEQ ID NO: 365) VL CDR2 RASNLDS RAS RASNLDS RAS LLIYRASNLD RASNLDS (SEQ ID NO: 418) (SEQ ID NO: 419) (SEQ ID NO: 418) (SEQ ID NO: 419) (SEQ ID NO: 420) (SEQ ID NO: 418) VL CDR3 QQSNEDPYT QQSNEDPYT QQSNEDPYT SNEDPY QQSNEDPY QQSNEDPYT (SEQ ID NO: 474) (SEQ ID NO: 474) (SEQ ID NO: 474) (SEQ ID NO: 475) (SEQ ID NO: 476) (SEQ ID NO: 474) VH Sequence: QVQLHQPGAELVKPGASVKLSCKTSGYTFTSYWMQWVKQRPGQGLEWIGEIDPSDSYTNYNQKFKGKATLTVDTSSTTAYMQLSSLTSEDSAVY YCARPLDRSAYYFDYWGQGTTLTVSS (SEQ ID NO: 37) VL Sequence: DIVLTQSPASLAVSLGQRATISCRASESVDFSGNSFMHWYQQKPGQPPKLLIYRASNLDSGIPARFSGVGSRTDFTLTINPVEADDVATYYCQQSN EDPYTFGGGTKLEIE (SEQ ID NO: 38)

TABLE 20 Antibody 2B11 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYSITSGYYWN GYSITSGYY SGYYWN GYSITSGY TSGYYWN GYSITSGYYWN Seq. (SEQ ID NO: 127) (SEQ ID NO: 128) (SEQ ID NO: 129) (SEQ ID NO: 130) (SEQ ID NO: 131) (SEQ ID NO: 127) VH CDR2 HIANDGSNYYNPF IANDGSN HIANDGSNYYNPF NDG WMGHIANDGSNY HIANDGSNY LKH (SEQ ID NO: 216) LKH (SEQ ID NO: 217) (SEQ ID NO: 218) (SEQ ID NO: 219) (SEQ ID NO: 215) (SEQ ID NO: 215) VH CDR3 GGSYFDYVDY ARGGSYFDYVDY GGSYFDYVDY GSYFDYVD ARGGSYFDYVD GGSYFDYVDY (SEQ ID NO: 293) (SEQ ID NO: 294) (SEQ ID NO: 293) (SEQ ID NO: 295) (SEQ ID NO: 296) (SEQ ID NO: 293) VL CDR VL CDR1 RASQDISNYLN QDISNY RASQDISNYLN SQDISNY SNYLNWY RASQDISNYLN Seq. (SEQ ID NO: 369) (SEQ ID NO: 370) (SEQ ID NO: 369) (SEQ ID NO: 371) (SEQ ID NO: 372) (SEQ ID NO: 369) VL CDR2 YTSRLHS YTS YTSRLHS YTS LLIYYTSRLH YTSRLHS (SEQ ID NO: 421) (SEQ ID NO: 405) (SEQ ID NO: 421) (SEQ ID NO: 405) (SEQ ID NO: 422) (SEQ ID NO: 421) VL CDR3 QQGNTLPFT QQGNTLPFT QQGNTLPFT GNTLPF QQGNTLPF QQGNTLPFT (SEQ ID NO: 477) (SEQ ID NO: 477) (SEQ ID NO: 477) (SEQ ID NO: 478) (SEQ ID NO: 479) (SEQ ID NO: 477) VH Sequence: DVQLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGHIANDGSNYYNPFLKHRVSITRDTSKNQFFLKLNSVTIQDTATYYC ARGGSYFDYVDYWGQGTTLTVSS (SEQ ID NO: 39) VL Sequence: DIQMTQTTSSLSASLGDRVTINCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTITNLEQEDIATYFCQQGNTLPFTF GSGTKLEIK (SEQ ID NO: 40)

TABLE 21 Antibody 1A3 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GFTFTDYYMN GFTFTDYY DYYMN GFTFTDY TDYYMN GFTFTDYYMN Seq. (SEQ ID NO: 488) (SEQ ID NO: 1795) (SEQ ID NO: 490) (SEQ ID NO: 1796) (SEQ ID NO: 491) (SEQ ID NO: 488) VH CDR2 DIIPNNGVTSYNQ IIPNNGVT DIIPNNGVTSYNQ PNNG WIGDIIPNNGVTS DIIPNNGVTS KFKG (SEQ ID NO: 498) KFKG (SEQ ID NO: 499) (SEQ ID NO: 500) (SEQ ID NO: 501) (SEQ ID NO: 497) (SEQ ID NO: 497) VH CDR3 EWLLRGMDY AREWLLRGMDY EWLLRGMDY WLLRGMD AREWLLRGMD EWLLRGMDY (SEQ ID NO: 515) (SEQ ID NO: 516) (SEQ ID NO: 515) (SEQ ID NO: 517) (SEQ ID NO: 518) (SEQ ID NO: 515) VL CDR VL CDR1 RSSKSLLHSNGIT KSLLHSNGITY RSSKSLLHSNGIT SKSLLHSNGITY LHSNGITYLYWY RSSKSLLHSNGIT Seq. YLY (SEQ ID NO: 532) YLY (SEQ ID NO: 533) (SEQ ID NO: 534) YLY (SEQ ID NO: 531) (SEQ ID NO: 531) (SEQ ID NO: 531) VL CDR2 QMSNLAS QMS QMSNLAS QMS LLIYQMSNLA QMSNLAS (SEQ ID NO: 547) (SEQ ID NO: 548) (SEQ ID NO: 547) (SEQ ID NO: 548) (SEQ ID NO: 549) (SEQ ID NO: 547) VL CDR3 AQHLELTWT AQHLELTWT AQHLELTWT HLELTW AQHLELTW AQHLELTWT (SEQ ID NO: 558) (SEQ ID NO: 558) (SEQ ID NO: 558) (SEQ ID NO: 559) (SEQ ID NO: 560) (SEQ ID NO: 558) VH Sequence: EVQLQQSGPELVKPGASVKISCKASGFTFTDYYMNWVKQSHGKSLEWIGDIIPNNGVTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYY CAREWLLRGMDYWGQGTSVTVSS (SEQ ID NO: 480) VL Sequence: DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQH LELTWTFGGGTKLEIK (SEQ ID NO: 481)

TABLE 22 Antibody P1B6 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTDYYMN GYTFTDYY DYYMN GYTFTDY TDYYMN GYTFTDYYMN Seq. (SEQ ID NO: 489) (SEQ ID NO: 57) (SEQ ID NO: 490) (SEQ ID NO: 49) (SEQ ID NO: 491) (SEQ ID NO: 489) VH CDR2 DINPNNGGPIYN INPNNGGP DINPNNGGPIYN PNNG WIGDINPNNGGPI DINPNNGGPI QKFKG (SEQ ID NO: 503) QKFKG (SEQ ID NO: 499) (SEQ ID NO: 504) (SEQ ID NO: 505) (SEQ ID NO: 502) (SEQ ID NO: 502) VH CDR3 SDSAWFTY ARSDSAWFTY SDSAWFTY DSAWFT ARSDSAWFT SDSAWFTY (SEQ ID NO: 519) (SEQ ID NO: 520) (SEQ ID NO: 519) (SEQ ID NO: 521) (SEQ ID NO: 522) (SEQ ID NO: 519) VL CDR VL CDR1 SASSSVSYMY SSVSY SASSSVSYMY SSSVSY SYMYWY SASSSVSYMY Seq. (SEQ ID NO: 535) (SEQ ID NO: 536) (SEQ ID NO: 535) (SEQ ID NO: 537) (SEQ ID NO: 538) (SEQ ID NO: 535) VL CDR2 DTSNLAS DTS DTSNLAS DTS LLIYDTSNLA DTSNLAS (SEQ ID NO: 550) (SEQ ID NO: 551) (SEQ ID NO: 550) (SEQ ID NO: 551) (SEQ ID NO: 552) (SEQ ID NO: 550) VL CDR3 QQWNSYPPT QQWNSYPPT QQWNSYPPT WNSYPP QQWNSYPP QQWNSYPPT (SEQ ID NO: 561) (SEQ ID NO: 561) (SEQ ID NO: 561) (SEQ ID NO: 562) (SEQ ID NO: 563) (SEQ ID NO: 561) VH Sequence: EVQLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQTHGKSLEWIGDINPNNGGPIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVY YCARSDSAWFTYWGQGTLVTVSA (SEQ ID NO: 482) VL Sequence: QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTFYSITISRMEAEDAATYYCQQWNSYPP TFGGGTKLEIK (SEQ ID NO: 483)

TABLE 23 Antibody P1H8 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GYTFTDYYMN GYTFTDYY DYYMN GYTFTDY TDYYMN GYTFTDYYMN Seq. (SEQ ID NO: 489) (SEQ ID NO: 57) (SEQ ID NO: 490) (SEQ ID NO: 49) (SEQ ID NO: 491) (SEQ ID NO: 489) VH CDR2 DINPNNGGTTYN INPNNGGT DINPNNGGTTYN PNNG WIGDINPNNGGT DINPNNGGTT QKFKG (SEQ ID NO: 507) QKFKG (SEQ ID NO: 499) T (SEQ ID NO: 509) (SEQ ID NO: 506) (SEQ ID NO: 506) (SEQ ID NO: 508) VH CDR3 QGPWYFDV ARQGPWYFDV QGPWYFDV GPWYFD ARQGPWYFD QGPWYFDV (SEQ ID NO: 523) (SEQ ID NO: 524) (SEQ ID NO: 523) (SEQ ID NO: 525) (SEQ ID NO: 526) (SEQ ID NO: 523) VL CDR VL CDR1 RSSQTIVHSNGY QTIVHSNGYTY RSSQTIVHSNGY SQTIVHSNGYTY VHSNGYTYLEWY RSSQTIVHSNGY Seq. TYLE (SEQ ID NO: 540) TYLE (SEQ ID NO: 541) (SEQ ID NO: 542) TYLE (SEQ ID NO: 539) (SEQ ID NO: 539) (SEQ ID NO: 539) VL CDR2 KVSNRFS KVS KVSNRFS KVS LLIYKVSNRF KVSNRFS (SEQ ID NO: 553) (SEQ ID NO: 554) (SEQ ID NO: 553) (SEQ ID NO: 554) (SEQ ID NO: 555) (SEQ ID NO: 553) VL CDR3 FQGSHVPWT FQGSHVPWT FQGSHVPWT QGSHVPW FQGSHVPW FQGSHVPWT (SEQ ID NO: 564) (SEQ ID NO: 564) (SEQ ID NO: 564) (SEQ ID NO: 565) (SEQ ID NO: 566) (SEQ ID NO: 564) VH Sequence: EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTTYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYY CARQGPWYFDVWGTGTTVTVSS (SEQ ID NO: 484) VL Sequence: DVLMTQTPLSLPVSLGDQASISCRSSQTIVHSNGYTYLEWYLQKPGQSPKWYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQG SHVPWTFGGGTKLEIK (SEQ ID NO: 485)

TABLE 24 Antibody P8G4 CDR Sequences Exemplary IMGT Kabat Chothia Contact AbM VH CDR VH CDR1 GFSLTSYGVH GFSLTSYG SYGVH GFSLTSY TSYGVH GFSLTSYGVH Seq. (SEQ ID NO: 492) (SEQ ID NO: 493) (SEQ ID NO: 494) (SEQ ID NO: 495) (SEQ ID NO: 496) (SEQ ID NO: 492) VH CDR2 VLWSGGSTDYN LWSGGST VLWSGGSTDYN GGS WLGVLWSGGST VLWSGGSTD AAFIS (SEQ ID NO: 511) AAFIS (SEQ ID NO: 512) D (SEQ ID NO: 514) (SEQ ID NO: 510) (SEQ ID NO: 510) (SEQ ID NO: 513) VH CDR3 NFGDY ARNFGDY NFGDY FGD ARNFGD NFGDY (SEQ ID NO: 527) (SEQ ID NO: 528) (SEQ ID NO: 527) (SEQ ID NO: 529) (SEQ ID NO: 530) (SEQ ID NO: 527) VL CDR VL CDR1 SASSRVSYMH SRVSY SASSRVSYMH SSRVSY SYMHWY SASSRVSYMH Seq. (SEQ ID NO: 543) (SEQ ID NO: 544) (SEQ ID NO: 543) (SEQ ID NO: 545) (SEQ ID NO: 546) (SEQ ID NO: 543) VL CDR2 DTSKLAS DTS DTSKLAS DTS RWIYDTSKLA DTSKLAS (SEQ ID NO: 556) (SEQ ID NO: 551) (SEQ ID NO: 556) (SEQ ID NO: 551) (SEQ ID NO: 557) (SEQ ID NO: 556) VL CDR3 QQWNNNPPT QQWNNNPPT QQWNNNPPT WNNNPP QQWNNNPP QQWNNNPPT (SEQ ID NO: 567) (SEQ ID NO: 567) (SEQ ID NO: 567) (SEQ ID NO: 568) (SEQ ID NO: 569) (SEQ ID NO: 567) VH Sequence: QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLDWLGVLWSGGSTDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYC ARNFGDYWGQGTSVTVSS (SEQ ID NO: 486) VL Sequence: QIVLTQSPAIMSASPGEKVTMTCSASSRVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWNNNP PTFGAGTTLELK (SEQ ID NO: 487)

In some embodiments, the antibodies provided herein comprise a VH region or VH domain In other embodiments, the antibodies provided herein comprise a VL region or VL chain. In some embodiments, the antibodies provided herein have a combination of (i) a VH domain or VH region; and/or (ii) a VL domain or VL region.

In some embodiments, an antibody provided herein comprises or consists of six CDRs, for example, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-24. In some embodiments, an antibody provided herein can comprise less than six CDRs. In some embodiments, the antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-24. In some embodiments, the antibody comprises or consists of one, two, three, four, or five CDRs selected from the group consisting of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 of the murine monoclonal antibody selected from the group consisting of: (a) the antibody designated 1C1; (b) the antibody designated 3P10; (c) the antibody designated 12A3; (d) the antibody designated 5F12; (e) the antibody designated 5A20; (f) the antibody designated 8D8; (g) the antibody designated 17J16; (h) t the antibody designated 25M22; (i) the antibody designated 2B8; (j) the antibody designated 22N5; (k) the antibody designated 2123; (l) the antibody designated 6N16; (m) the antibody designated 1B3; (n) the antibody designated 19K19; (o) the antibody designated 2B3; (p) the antibody designated 8C10; (q) the antibody designated 2A9; (r) the antibody designated 24G2; (s) the antibody designated 6G9; (t) the antibody designated 2B111; (u) the antibody designated 1A3; (v) the antibody designated P1B6; (w) the antibody designated P1H8; or (x) the antibody designated P8G4 described herein. Accordingly, in some embodiments, the antibody comprises or consists of one, two, three four or five CDRs of anyone of the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and/or VL CDR3 identified in Tables 1-24.

In some embodiments, the antibodies provided herein comprise or consist of one or more (e.g., one, two or three) VH CDRs listed in Tables 1-24. In other embodiments, the antibodies provided herein comprise one or more (e.g., one, two or three) VL CDRs listed in Tables 1-24. In yet other embodiments, the antibodies provided herein comprise one or more (e.g., one, two or three) VH CDRs listed in Tables 1-24 and one or more VL CDRs listed in Tables 1-24. Accordingly, in certain embodiments, the antibodies comprise a VH CDR1 having the amino acid sequence of any one of SEQ ID NOS: 57, 49, 57, 49, 221-296, 515-530, 488-493, 1795, 1796, 488-493, 1795, 1796. In another embodiment, the antibodies comprise a VH CDR2 having the amino acid sequence of any one of SEQ ID NOS: 132-220, 497-514. In another embodiment, the antibodies comprise a VH CDR3 having the amino acid sequence of any one of SEQ ID NOS: 57, 49, 57, 49, 221-296, 515-530, 488-493, 1795, 1796, 488-493, 1795, 1796. In certain embodiments, the antibodies comprise a VH CDR1 and/or a VH CDR2 and/or a VH CDR3 independently selected from a VH CDR1, VH CDR2, VH CDR3 as depicted in any one of the amino acid sequences depicted in Table 1-24. In certain embodiments, the antibodies comprise a VL CDR1 having the amino acid sequence of any one of SEQ ID NOS: 297-372, 531-546. In another embodiment, the antibodies comprise a VL CDR2 having the amino acid sequence of any one of SEQ ID NOS: 373-422. In another embodiment, the antibodies comprise a VL CDR3 having the amino acid sequence of any one of SEQ ID NOS: 423-479, 558-569. In certain embodiments, the antibodies comprise a VL CDR1 and/or a VL CDR2 and/or a VL CDR3 independently selected from a VL CDR1, VL CDR2, VL CDR3 as depicted in any one of the amino acid sequences depicted in Tables 1-24.

Also provided herein are antibodies comprising one or more (e.g., one, two or three) VH CDRs and one or more (e.g., one, two or three) VL CDRs listed in Tables 1-24. In particular, provided herein is an antibody comprising: a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR1 (SEQ ID NOS: 297-372, 531-546); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR1 (SEQ ID NOS: 297-372, 531-546); a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR1 (SEQ ID NOS: 297-372, 531-546); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR1 (SEQ ID NOS: 297-372, 531-546); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR1 (SEQ ID NOS: 297-372, 531-546), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR1 (SEQ ID NOS: 297-372, 531-546); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR2 (SEQ ID NOS: 373-422); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR2 (SEQ ID NOS: 373-422) and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VL CDR1 (SEQ ID NOS: 297-372, 531-546), a VL CDR2 (SEQ ID NOS: 373-422), and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR1 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546), a VL CDR2 (SEQ ID NOS: 373-422), and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); a VH CDR2 (SEQ ID NOS: 132-220, 497-514), a VH CDR3 (SEQ ID NOS: 57, 49, 221-296, 515-530, 488-493, 1795, 1796), a VL CDR1 (SEQ ID NOS: 297-372, 531-546), a VL CDR2 (SEQ ID NOS: 373-422), and a VL CDR3 (SEQ ID NOS: 423-479, 558-569); or any combination thereof of the VH CDRs (SEQ ID NOS: 41-296) and VL CDRs (SEQ ID NOS: 297-477) listed in Tables 1-24.

In certain embodiments, an antibody or fragment thereof described herein comprises a humanized framework region (FR) sequence. In certain embodiments, an antibody or fragment thereof described herein comprises a VH region comprising a VH FR1, a VH FR2, a VH FR3 and a VH FR4 amino acid sequence depicted in Table 25; and/or (b) a VL region comprising a VL FR1, a VL FR2, a VL FR3 and a VL FR4 amino acid sequence depicted in Table 25.

TABLE 25 Exemplary Framework Sequences for Humanized Anti-GFRAL Antibodies VH Clones Humanized SEQ ID NO: VH Framework 1 (FR1) QVQLQESGPGLVKPSETLSLTCTVS 1C1 570 QMQLQESGPGLVKPSETLSLICTVS 1C1 571 QVQLVQSGAEVKKPGSSVKVSCKAS 3P10, 5F12, 25M22, 572 17J16 QIQLVQSGAEVKKPGSSVKVSCKAS 3P10 573 QVQLVQSGAEVKKPGATVKISCKVS 3P10 574 QIQLVQSGAEVKKPGATVKISCKVS 3P10 575 QVQLVQSGAEVVKPGSSVKVSCKAS 5F12 576 QVQLVQSGAEVKKPGASVKVSCKAS 5F12 577 EVQLVQSGAEVKKPGESLKISCKGS 25M22 578 VH Framework 2 (FR2) WIRQPPGKGLEWIG 1C1 579 WIRQPPGKGLEWLG 1C1 580 WVRQAPGQGLEWMG 3P10, 5F12, 25M22, 581 17J16 WVRQAPGKGLEWMG 3P10 582 WVRQAPGQALEWMG 3P10 583 WVRQAPGQGLKWMG 3P10 584 WVRQAPGKALEWMG 3P10 585 WVRQAPGKGLKWMG 3P10 586 WVRQAPGQALKWMG 3P10 587 WVRQAPGKALKWMG 3P10 588 WVKQAPGQGLEWIG 5F12, 17J16 589 WVRQAPGQGLEWIG 5F12, 25M22, 17J16 590 WVRQAPGQGLEWIA 5F12 591 WVKQAPGQGLEWIG 5F12, 17J16 592 WVKQAPGQGLEWIA 5F12 593 WVRQAPGQGLEWIA 5F12 594 WVKQAPGQGLEWIA 5F12 595 WMQQAPGKGLEWIG 3P10 596 WVRQAPGQRLEWMG 5F12 597 WVRQAPGQRLEWIG 5F12 598 WVRQAPGQRLEWMA 5F12 599 WVRQAPGQRLEWIA 5F12 600 WVRQMPGKGLEWMG 25M22 601 WVRQMPGKGLEWIG 25M22 602 VH Framework 3 (FR3) RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 603 RLTISVDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 604 RVTISKDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 605 RVTISVDNSKNQFSLKLSSVTAADTAVYYCAR 1C1 606 RVTISVDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 607 RVTISVDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 608 RVTISVDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 609 RVTISVDTSKNQFSLKMSSVTAADTAVYYCAR 1C1 610 RVTISVDTSKNQFSLKLSSLTAADTAVYYCAR 1C1 611 RVTISVDTSKNQFSLKLSSVQAADTAVYYCAR 1C1 612 RVTISVDTSKNQFSLKLSSVTAQDTAVYYCAR 1C1 613 RLTISKDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 614 RLTISVDNSKNQFSLKLSSVTAADTAVYYCAR 1C1 615 RLTISVDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 616 RLTISVDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 617 RLTISVDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 618 RLTISVDTSKNQFSLKMSSVTAADTAVYYCAR 1C1 619 RLTISVDTSKNQFSLKLSSLTAADTAVYYCAR 1C1 620 RLTISVDTSKNQFSLKLSSVQAADTAVYYCAR 1C1 621 RLTISVDTSKNQFSLKLSSVTAQDTAVYYCAR 1C1 622 RVTISKDNSKNQFSLKLSSVTAADTAVYYCAR 1C1 623 RVTISKDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 624 RVTISKDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 625 RVTISKDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 626 RVTISKDTSKNQFSLKMSSVTAADTAVYYCAR 1C1 627 RVTISKDTSKNQFSLKLSSLTAADTAVYYCAR 1C1 628 RVTISKDTSKNQFSLKLSSVQAADTAVYYCAR 1C1 629 RVTISKDTSKNQFSLKLSSVTAQDTAVYYCAR 1C1 630 RVTISVDNSKSQFSLKLSSVTAADTAVYYCAR 1C1 631 RVTISVDNSKNQVSLKLSSVTAADTAVYYCAR 1C1 632 RVTISVDNSKNQFSFKLSSVTAADTAVYYCAR 1C1 633 RVTISVDNSKNQFSLKMSSVTAADTAVYYCAR 1C1 634 RVTISVDNSKNQFSLKLSSLTAADTAVYYCAR 1C1 635 RVTISVDNSKNQFSLKLSSVQAADTAVYYCAR 1C1 636 RVTISVDNSKNQFSLKLSSVTAQDTAVYYCAR 1C1 637 RVTISVDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 638 RVTISVDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 639 RVTISVDTSKSQFSLKMSSVTAADTAVYYCAR 1C1 640 RVTISVDTSKSQFSLKLSSLTAADTAVYYCAR 1C1 641 RVTISVDTSKSQFSLKLSSVQAADTAVYYCAR 1C1 642 RVTISVDTSKSQFSLKLSSVTAQDTAVYYCAR 1C1 643 RVTISVDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 644 RVTISVDTSKNQVSLKMSSVTAADTAVYYCAR 1C1 645 RVTISVDTSKNQVSLKLSSLTAADTAVYYCAR 1C1 646 RVTISVDTSKNQVSLKLSSVQAADTAVYYCAR 1C1 647 RVTISVDTSKNQVSLKLSSVTAQDTAVYYCAR 1C1 648 RVTISVDTSKNQFSFKMSSVTAADTAVYYCAR 1C1 649 RVTISVDTSKNQFSFKLSSLTAADTAVYYCAR 1C1 650 RVTISVDTSKNQFSFKLSSVQAADTAVYYCAR 1C1 651 RVTISVDTSKNQFSFKLSSVTAQDTAVYYCAR 1C1 652 RVTISVDTSKNQFSLKMSSLTAADTAVYYCAR 1C1 653 RVTISVDTSKNQFSLKMSSVQAADTAVYYCAR 1C1 654 RVTISVDTSKNQFSLKMSSVTAQDTAVYYCAR 1C1 655 RVTISVDTSKNQFSLKLSSLQAADTAVYYCAR 1C1 656 RVTISVDTSKNQFSLKLSSLTAQDTAVYYCAR 1C1 657 RVTISVDTSKNQFSLKLSSVQAQDTAVYYCAR 1C1 658 RLTISKDNSKNQFSLKLSSVTAADTAVYYCAR 1C1 659 RLTISKDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 660 RLTISKDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 661 RLTISKDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 662 RLTISKDTSKNQFSLKMSSVTAADTAVYYCAR 1C1 663 RLTISKDTSKNQFSLKLSSLTAADTAVYYCAR 1C1 664 RLTISKDTSKNQFSLKLSSVQAADTAVYYCAR 1C1 665 RLTISKDTSKNQFSLKLSSVTAQDTAVYYCAR 1C1 666 RLTISVDNSKSQFSLKLSSVTAADTAVYYCAR 1C1 667 RLTISVDNSKNQVSLKLSSVTAADTAVYYCAR 1C1 668 RLTISVDNSKNQFSFKLSSVTAADTAVYYCAR 1C1 669 RLTISVDNSKNQFSLKMSSVTAADTAVYYCAR 1C1 670 RLTISVDNSKNQFSLKLSSLTAADTAVYYCAR 1C1 671 RLTISVDNSKNQFSLKLSSVQAADTAVYYCAR 1C1 672 RLTISVDNSKNQFSLKLSSVTAQDTAVYYCAR 1C1 673 RLTISVDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 674 RLTISVDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 675 RLTISVDTSKSQFSLKMSSVTAADTAVYYCAR 1C1 676 RLTISVDTSKSQFSLKLSSLTAADTAVYYCAR 1C1 677 RLTISVDTSKSQFSLKLSSVQAADTAVYYCAR 1C1 678 RLTISVDTSKSQFSLKLSSVTAQDTAVYYCAR 1C1 679 RLTISVDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 680 RLTISVDTSKNQVSLKMSSVTAADTAVYYCAR 1C1 681 RLTISVDTSKNQVSLKLSSLTAADTAVYYCAR 1C1 682 RLTISVDTSKNQVSLKLSSVQAADTAVYYCAR 1C1 683 RLTISVDTSKNQVSLKLSSVTAQDTAVYYCAR 1C1 684 RLTISVDTSKNQFSFKMSSVTAADTAVYYCAR 1C1 685 RLTISVDTSKNQFSFKLSSLTAADTAVYYCAR 1C1 686 RLTISVDTSKNQFSFKLSSVQAADTAVYYCAR 1C1 687 RLTISVDTSKNQFSFKLSSVTAQDTAVYYCAR 1C1 688 RLTISVDTSKNQFSLKMSSLTAADTAVYYCAR 1C1 689 RLTISVDTSKNQFSLKMSSVQAADTAVYYCAR 1C1 690 RLTISVDTSKNQFSLKMSSVTAQDTAVYYCAR 1C1 691 RLTISVDTSKNQFSLKLSSLQAADTAVYYCAR 1C1 692 RLTISVDTSKNQFSLKLSSLTAQDTAVYYCAR 1C1 693 RLTISVDTSKNQFSLKLSSVQAQDTAVYYCAR 1C1 694 RVTISKDNSKSQFSLKLSSVTAADTAVYYCAR 1C1 695 RVTISKDNSKNQVSLKLSSVTAADTAVYYCAR 1C1 696 RVTISKDNSKNQFSFKLSSVTAADTAVYYCAR 1C1 697 RVTISKDNSKNQFSLKMSSVTAADTAVYYCAR 1C1 698 RVTISKDNSKNQFSLKLSSLTAADTAVYYCAR 1C1 699 RVTISKDNSKNQFSLKLSSVQAADTAVYYCAR 1C1 700 RVTISKDNSKNQFSLKLSSVTAQDTAVYYCAR 1C1 701 RVTISKDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 702 RVTISKDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 703 RVTISKDTSKSQFSLKMSSVTAADTAVYYCAR 1C1 704 RVTISKDTSKSQFSLKLSSLTAADTAVYYCAR 1C1 705 RVTISKDTSKSQFSLKLSSVQAADTAVYYCAR 1C1 706 RVTISKDTSKSQFSLKLSSVTAQDTAVYYCAR 1C1 707 RVTISKDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 708 RVTISKDTSKNQVSLKMSSVTAADTAVYYCAR 1C1 709 RVTISKDTSKNQVSLKLSSLTAADTAVYYCAR 1C1 710 RVTISKDTSKNQVSLKLSSVQAADTAVYYCAR 1C1 711 RVTISKDTSKNQVSLKLSSVTAQDTAVYYCAR 1C1 712 RVTISKDTSKNQFSFKMSSVTAADTAVYYCAR 1C1 713 RVTISKDTSKNQFSFKLSSLTAADTAVYYCAR 1C1 714 RVTISKDTSKNQFSFKLSSVQAADTAVYYCAR 1C1 715 RVTISKDTSKNQFSFKLSSVTAQDTAVYYCAR 1C1 716 RVTISKDTSKNQFSLKMSSLTAADTAVYYCAR 1C1 717 RVTISKDTSKNQFSLKMSSVQAADTAVYYCAR 1C1 718 RVTISKDTSKNQFSLKMSSVTAQDTAVYYCAR 1C1 719 RVTISKDTSKNQFSLKLSSLQAADTAVYYCAR 1C1 720 RVTISKDTSKNQFSLKLSSLTAQDTAVYYCAR 1C1 721 RVTTSKDTSKNQFSLKLSSVQAQDTAVYYCAR 1C1 722 RVTISVDNSKSQVSLKLSSVTAADTAVYYCAR 1C1 723 RVTISVDNSKSQFSFKLSSVTAADTAVYYCAR 1C1 724 RVTISVDNSKSQFSLKMSSVTAADTAVYYCAR 1C1 725 RVTISVDNSKSQFSLKLSSLTAADTAVYYCAR 1C1 726 RVTISVDNSKSQFSLKLSSVQAADTAVYYCAR 1C1 727 RVTISVDNSKSQFSLKLSSVTAQDTAVYYCAR 1C1 728 RVTISVDNSKNQVSFKLSSVTAADTAVYYCAR 1C1 729 RVTISVDNSKNQVSLKMSSVTAADTAVYYCAR 1C1 730 RVTISVDNSKNQVSLKLSSLTAADTAVYYCAR 1C1 731 RVTISVDNSKNQVSLKLSSVQAADTAVYYCAR 1C1 732 RVTISVDNSKNQVSLKLSSVTAQDTAVYYCAR 1C1 733 RVTISVDNSKNQFSFKMSSVTAADTAVYYCAR 1C1 734 RVTISVDNSKNQFSFKLSSLTAADTAVYYCAR 1C1 735 RVTISVDNSKNQFSFKLSSVQAADTAVYYCAR 1C1 736 RVTISVDNSKNQFSFKLSSVTAQDTAVYYCAR 1C1 737 RVTISVDNSKNQFSLKMSSLTAADTAVYYCAR 1C1 738 RVTISVDNSKNQFSLKMSSVQAADTAVYYCAR 1C1 739 RVTISVDNSKNQFSLKMSSVTAQDTAVYYCAR 1C1 740 RVTISVDNSKNQFSLKLSSLQAADTAVYYCAR 1C1 741 RVTISVDNSKNQFSLKLSSLTAQDTAVYYCAR 1C1 742 RVTISVDNSKNQFSLKLSSVQAQDTAVYYCAR 1C1 743 RVTISVDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 744 RVTISVDTSKSQVSLKMSSVTAADTAVYYCAR 1C1 745 RVTISVDTSKSQVSLKLSSLTAADTAVYYCAR 1C1 746 RVTISVDTSKSQVSLKLSSVQAADTAVYYCAR 1C1 747 RVTISVDTSKSQVSLKLSSVTAQDTAVYYCAR 1C1 748 RVTISVDTSKSQFSFKMSSVTAADTAVYYCAR 1C1 749 RVTISVDTSKSQFSFKLSSLTAADTAVYYCAR 1C1 750 RVTTSVDTSKSQFSFKLSSVQAADTAVYYCAR 1C1 751 RVTISVDTSKSQFSFKLSSVTAQDTAVYYCAR 1C1 752 RVTISVDTSKSQFSLKMSSLTAADTAVYYCAR 1C1 753 RVTISVDTSKSQFSLKMSSVQAADTAVYYCAR 1C1 754 RVTISVDTSKSQFSLKMSSVTAQDTAVYYCAR 1C1 755 RVTISVDTSKSQFSLKLSSLQAADTAVYYCAR 1C1 756 RVTISVDTSKSQFSLKLSSLTAQDTAVYYCAR 1C1 757 RVTISVDTSKSQFSLKLSSVQAQDTAVYYCAR 1C1 758 RVTISVDTSKNQVSFKMSSVTAADTAVYYCAR 1C1 759 RVTISVDTSKNQVSFKLSSLTAADTAVYYCAR 1C1 760 RVTISVDTSKNQVSFKLSSVQAADTAVYYCAR 1C1 761 RVTISVDTSKNQVSFKLSSVTAQDTAVYYCAR 1C1 762 RVTISVDTSKNQVSLKMSSLTAADTAVYYCAR 1C1 763 RVTISVDTSKNQVSLKMSSVQAADTAVYYCAR 1C1 764 RVTISVDTSKNQVSLKMSSVTAQDTAVYYCAR 1C1 765 RVTISVDTSKNQVSLKLSSLQAADTAVYYCAR 1C1 766 RVTISVDTSKNQVSLKLSSLTAQDTAVYYCAR 1C1 767 RVTISVDTSKNQVSLKLSSVQAQDTAVYYCAR 1C1 768 RVTISVDTSKNQFSFKMSSLTAADTAVYYCAR 1C1 769 RVTISVDTSKNQFSFKMSSVQAADTAVYYCAR 1C1 770 RVTISVDTSKNQFSFKMSSVTAQDTAVYYCAR 1C1 771 RVTISVDTSKNQFSFKLSSLQAADTAVYYCAR 1C1 772 RVTISVDTSKNQFSFKLSSLTAQDTAVYYCAR 1C1 773 RVTISVDTSKNQFSFKLSSVQAQDTAVYYCAR 1C1 774 RVTISVDTSKNQFSLKMSSLQAADTAVYYCAR 1C1 775 RVTISVDTSKNQFSLKMSSLTAQDTAVYYCAR 1C1 776 RVTISVDTSKNQFSLKMSSVQAQDTAVYYCAR 1C1 777 RVTISVDTSKNQFSLKLSSLQAQDTAVYYCAR 1C1 778 RLTISKDNSKSQFSLKLSSVTAADTAVYYCAR 1C1 779 RLTISKDNSKNQVSLKLSSVTAADTAVYYCAR 1C1 780 RLTISKDNSKNQFSFKLSSVTAADTAVYYCAR 1C1 781 RLTISKDNSKNQFSLKMSSVTAADTAVYYCAR 1C1 782 RLTISKDNSKNQFSLKLSSLTAADTAVYYCAR 1C1 783 RLTISKDNSKNQFSLKLSSVQAADTAVYYCAR 1C1 784 RLTISKDNSKNQFSLKLSSVTAQDTAVYYCAR 1C1 785 RLTISKDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 786 RLTISKDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 787 RLTISKDTSKSQFSLKMSSVTAADTAVYYCAR 1C1 788 RLTISKDTSKSQFSLKLSSLTAADTAVYYCAR 1C1 789 RLTISKDTSKSQFSLKLSSVQAADTAVYYCAR 1C1 790 RLTISKDTSKSQFSLKLSSVTAQDTAVYYCAR 1C1 791 RLTISKDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 792 RLTISKDTSKNQVSLKMSSVTAADTAVYYCAR 1C1 793 RLTISKDTSKNQVSLKLSSLTAADTAVYYCAR 1C1 794 RLTISKDTSKNQVSLKLSSVQAADTAVYYCAR 1C1 795 RLTISKDTSKNQVSLKLSSVTAQDTAVYYCAR 1C1 796 RLTISKDTSKNQFSFKMSSVTAADTAVYYCAR 1C1 797 RLTISKDTSKNQFSFKLSSLTAADTAVYYCAR 1C1 798 RLTISKDTSKNQFSFKLSSVQAADTAVYYCAR 1C1 799 RLTISKDTSKNQFSFKLSSVTAQDTAVYYCAR 1C1 800 RLTISKDTSKNQFSLKMSSLTAADTAVYYCAR 1C1 801 RLTISKDTSKNQFSLKMSSVQAADTAVYYCAR 1C1 802 RLTISKDTSKNQFSLKMSSVTAQDTAVYYCAR 1C1 803 RLTISKDTSKNQFSLKLSSLQAADTAVYYCAR 1C1 804 RLTISKDTSKNQFSLKLSSLTAQDTAVYYCAR 1C1 805 RLTISKDTSKNQFSLKLSSVQAQDTAVYYCAR 1C1 806 RLTISVDNSKSQVSLKLSSVTAADTAVYYCAR 1C1 807 RLTISVDNSKSQFSFKLSSVTAADTAVYYCAR 1C1 808 RLTISVDNSKSQFSLKMSSVTAADTAVYYCAR 1C1 809 RLTISVDNSKSQFSLKLSSLTAADTAVYYCAR 1C1 810 RLTISVDNSKSQFSLKLSSVQAADTAVYYCAR 1C1 811 RLTISVDNSKSQFSLKLSSVTAQDTAVYYCAR 1C1 812 RLTISVDNSKNQVSFKLSSVTAADTAVYYCAR 1C1 813 RLTISVDNSKNQVSLKMSSVTAADTAVYYCAR 1C1 814 RLTISVDNSKNQVSLKLSSLTAADTAVYYCAR 1C1 815 RLTISVDNSKNQVSLKLSSVQAADTAVYYCAR 1C1 816 RLTISVDNSKNQVSLKLSSVTAQDTAVYYCAR 1C1 817 RLTISVDNSKNQFSFKMSSVTAADTAVYYCAR 1C1 818 RLTISVDNSKNQFSFKLSSLTAADTAVYYCAR 1C1 819 RLTISVDNSKNQFSFKLSSVQAADTAVYYCAR 1C1 820 RLTISVDNSKNQFSFKLSSVTAQDTAVYYCAR 1C1 821 RLTISVDNSKNQFSLKMSSLTAADTAVYYCAR 1C1 822 RLTISVDNSKNQFSLKMSSVQAADTAVYYCAR 1C1 823 RLTISVDNSKNQFSLKMSSVTAQDTAVYYCAR 1C1 824 RLTISVDNSKNQFSLKLSSLQAADTAVYYCAR 1C1 825 RLTISVDNSKNQFSLKLSSLTAQDTAVYYCAR 1C1 826 RLTISVDNSKNQFSLKLSSVQAQDTAVYYCAR 1C1 827 RLTISVDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 828 RLTISVDTSKSQVSLKMSSVTAADTAVYYCAR 1C1 829 RLTISVDTSKSQVSLKLSSLTAADTAVYYCAR 1C1 830 RLTISVDTSKSQVSLKLSSVQAADTAVYYCAR 1C1 831 RLTISVDTSKSQVSLKLSSVTAQDTAVYYCAR 1C1 832 RLTISVDTSKSQFSFKMSSVTAADTAVYYCAR 1C1 833 RLTISVDTSKSQFSFKLSSLTAADTAVYYCAR 1C1 834 RLTISVDTSKSQFSFKLSSVQAADTAVYYCAR 1C1 835 RLTISVDTSKSQFSFKLSSVTAQDTAVYYCAR 1C1 836 RLTISVDTSKSQFSLKMSSLTAADTAVYYCAR 1C1 837 RLTISVDTSKSQFSLKMSSVQAADTAVYYCAR 1C1 838 RLTISVDTSKSQFSLKMSSVTAQDTAVYYCAR 1C1 839 RLTISVDTSKSQFSLKLSSLQAADTAVYYCAR 1C1 840 RLTISVDTSKSQFSLKLSSLTAQDTAVYYCAR 1C1 841 RLTISVDTSKSQFSLKLSSVQAQDTAVYYCAR 1C1 842 RLTISVDTSKNQVSFKMSSVTAADTAVYYCAR 1C1 843 RLTISVDTSKNQVSFKLSSLTAADTAVYYCAR 1C1 844 RLTISVDTSKNQVSFKLSSVQAADTAVYYCAR 1C1 845 RLTISVDTSKNQVSFKLSSVTAQDTAVYYCAR 1C1 846 RLTISVDTSKNQVSLKMSSLTAADTAVYYCAR 1C1 847 RLTISVDTSKNQVSLKMSSVQAADTAVYYCAR 1C1 848 RLTISVDTSKNQVSLKMSSVTAQDTAVYYCAR 1C1 849 RLTISVDTSKNQVSLKLSSLQAADTAVYYCAR 1C1 850 RLTISVDTSKNQVSLKLSSLTAQDTAVYYCAR 1C1 851 RLTISVDTSKNQVSLKLSSVQAQDTAVYYCAR 1C1 852 RLTISVDTSKNQFSFKMSSLTAADTAVYYCAR 1C1 853 RLTISVDTSKNQFSFKMSSVQAADTAVYYCAR 1C1 854 RLTISVDTSKNQFSFKMSSVTAQDTAVYYCAR 1C1 855 RLTISVDTSKNQFSFKLSSLQAADTAVYYCAR 1C1 856 RLTISVDTSKNQFSFKLSSLTAQDTAVYYCAR 1C1 857 RLTISVDTSKNQFSFKLSSVQAQDTAVYYCAR 1C1 858 RLTISVDTSKNQFSLKMSSLQAADTAVYYCAR 1C1 859 RLTISVDTSKNQFSLKMSSLTAQDTAVYYCAR 1C1 860 RLTISVDTSKNQFSLKMSSVQAQDTAVYYCAR 1C1 861 RLTISVDTSKNQFSLKLSSLQAQDTAVYYCAR 1C1 862 RVTISKDNSKSQVSLKLSSVTAADTAVYYCAR 1C1 863 RVTISKDNSKSQFSFKLSSVTAADTAVYYCAR 1C1 864 RVTISKDNSKSQFSLKMSSVTAADTAVYYCAR 1C1 865 RVTISKDNSKSQFSLKLSSLTAADTAVYYCAR 1C1 866 RVTTSKDNSKSQFSLKLSSVQAADTAVYYCAR 1C1 867 RVTISKDNSKSQFSLKLSSVTAQDTAVYYCAR 1C1 868 RVTISKDNSKNQVSFKLSSVTAADTAVYYCAR 1C1 869 RVTISKDNSKNQVSLKMSSVTAADTAVYYCAR 1C1 870 RVTISKDNSKNQVSLKLSSLTAADTAVYYCAR 1C1 871 RVTISKDNSKNQVSLKLSSVQAADTAVYYCAR 1C1 872 RVTISKDNSKNQVSLKLSSVTAQDTAVYYCAR 1C1 873 RVTISKDNSKNQFSFKMSSVTAADTAVYYCAR 1C1 874 RVTISKDNSKNQFSFKLSSLTAADTAVYYCAR 1C1 875 RVTISKDNSKNQFSFKLSSVQAADTAVYYCAR 1C1 876 RVTISKDNSKNQFSFKLSSVTAQDTAVYYCAR 1C1 877 RVTISKDNSKNQFSLKMSSLTAADTAVYYCAR 1C1 878 RVTISKDNSKNQFSLKMSSVQAADTAVYYCAR 1C1 879 RVTISKDNSKNQFSLKMSSVTAQDTAVYYCAR 1C1 880 RVTISKDNSKNQFSLKLSSLQAADTAVYYCAR 1C1 881 RVTISKDNSKNQFSLKLSSLTAQDTAVYYCAR 1C1 882 RVTISKDNSKNQFSLKLSSVQAQDTAVYYCAR 1C1 883 RVTISKDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 884 RVTISKDTSKSQVSLKMSSVTAADTAVYYCAR 1C1 885 RVTISKDTSKSQVSLKLSSLTAADTAVYYCAR 1C1 886 RVTISKDTSKSQVSLKLSSVQAADTAVYYCAR 1C1 887 RVTISKDTSKSQVSLKLSSVTAQDTAVYYCAR 1C1 888 RVTISKDTSKSQFSFKMSSVTAADTAVYYCAR 1C1 889 RVTISKDTSKSQFSFKLSSLTAADTAVYYCAR 1C1 890 RVTISKDTSKSQFSFKLSSVQAADTAVYYCAR 1C1 891 RVTISKDTSKSQFSFKLSSVTAQDTAVYYCAR 1C1 892 RVTISKDTSKSQFSLKMSSLTAADTAVYYCAR 1C1 893 RVTISKDTSKSQFSLKMSSVQAADTAVYYCAR 1C1 894 RVTISKDTSKSQFSLKMSSVTAQDTAVYYCAR 1C1 895 RVTISKDTSKSQFSLKLSSLQAADTAVYYCAR 1C1 896 RVTISKDTSKSQFSLKLSSLTAQDTAVYYCAR 1C1 897 RVTISKDTSKSQFSLKLSSVQAQDTAVYYCAR 1C1 898 RVTISKDTSKNQVSFKMSSVTAADTAVYYCAR 1C1 899 RVTISKDTSKNQVSFKLSSLTAADTAVYYCAR 1C1 900 RVTISKDTSKNQVSFKLSSVQAADTAVYYCAR 1C1 901 RVTISKDTSKNQVSFKLSSVTAQDTAVYYCAR 1C1 902 RVTISKDTSKNQVSLKMSSLTAADTAVYYCAR 1C1 903 RVTISKDTSKNQVSLKMSSVQAADTAVYYCAR 1C1 904 RVTISKDTSKNQVSLKMSSVTAQDTAVYYCAR 1C1 905 RVTISKDTSKNQVSLKLSSLQAADTAVYYCAR 1C1 906 RVTISKDTSKNQVSLKLSSLTAQDTAVYYCAR 1C1 907 RVTISKDTSKNQVSLKLSSVQAQDTAVYYCAR 1C1 908 RVTISKDTSKNQFSFKMSSLTAADTAVYYCAR 1C1 909 RVTISKDTSKNQFSFKMSSVQAADTAVYYCAR 1C1 910 RVTISKDTSKNQFSFKMSSVTAQDTAVYYCAR 1C1 911 RVTISKDTSKNQFSFKLSSLQAADTAVYYCAR 1C1 912 RVTISKDTSKNQFSFKLSSLTAQDTAVYYCAR 1C1 913 RVTISKDTSKNQFSFKLSSVQAQDTAVYYCAR 1C1 914 RVTISKDTSKNQFSLKMSSLQAADTAVYYCAR 1C1 915 RVTISKDTSKNQFSLKMSSLTAQDTAVYYCAR 1C1 916 RVTISKDTSKNQFSLKMSSVQAQDTAVYYCAR 1C1 917 RVTISKDTSKNQFSLKLSSLQAQDTAVYYCAR 1C1 918 RVTISVDNSKSQVSFKLSSVTAADTAVYYCAR 1C1 919 RVTISVDNSKSQVSLKMSSVTAADTAVYYCAR 1C1 920 RVTISVDNSKSQVSLKLSSLTAADTAVYYCAR 1C1 921 RVTISVDNSKSQVSLKLSSVQAADTAVYYCAR 1C1 922 RVTISVDNSKSQVSLKLSSVTAQDTAVYYCAR 1C1 923 RVTISVDNSKSQFSFKMSSVTAADTAVYYCAR 1C1 924 RVTTSVDNSKSQFSFKLSSLTAADTAVYYCAR 1C1 925 RVTISVDNSKSQFSFKLSSVQAADTAVYYCAR 1C1 926 RVTISVDNSKSQFSFKLSSVTAQDTAVYYCAR 1C1 927 RVTISVDNSKSQFSLKMSSLTAADTAVYYCAR 1C1 928 RVTISVDNSKSQFSLKMSSVQAADTAVYYCAR 1C1 929 RVTISVDNSKSQFSLKMSSVTAQDTAVYYCAR 1C1 930 RVTISVDNSKSQFSLKLSSLQAADTAVYYCAR 1C1 931 RVTISVDNSKSQFSLKLSSLTAQDTAVYYCAR 1C1 932 RVTISVDNSKSQFSLKLSSVQAQDTAVYYCAR 1C1 933 RVTISVDNSKNQVSFKMSSVTAADTAVYYCAR 1C1 934 RVTISVDNSKNQVSFKLSSLTAADTAVYYCAR 1C1 935 RVTISVDNSKNQVSFKLSSVQAADTAVYYCAR 1C1 936 RVTISVDNSKNQVSFKLSSVTAQDTAVYYCAR 1C1 937 RVTISVDNSKNQVSLKMSSLTAADTAVYYCAR 1C1 938 RVTISVDNSKNQVSLKMSSVQAADTAVYYCAR 1C1 939 RVTISVDNSKNQVSLKMSSVTAQDTAVYYCAR 1C1 940 RVTISVDNSKNQVSLKLSSLQAADTAVYYCAR 1C1 941 RVTISVDNSKNQVSLKLSSLTAQDTAVYYCAR 1C1 942 RVTISVDNSKNQVSLKLSSVQAQDTAVYYCAR 1C1 943 RVTISVDNSKNQFSFKMSSLTAADTAVYYCAR 1C1 944 RVTISVDNSKNQFSFKMSSVQAADTAVYYCAR 1C1 945 RVTISVDNSKNQFSFKMSSVTAQDTAVYYCAR 1C1 946 RVTISVDNSKNQFSFKLSSLQAADTAVYYCAR 1C1 947 RVTISVDNSKNQFSFKLSSLTAQDTAVYYCAR 1C1 948 RVTISVDNSKNQFSFKLSSVQAQDTAVYYCAR 1C1 949 RVTISVDNSKNQFSLKMSSLQAADTAVYYCAR 1C1 950 RVTISVDNSKNQFSLKMSSLTAQDTAVYYCAR 1C1 951 RVTISVDNSKNQFSLKMSSVQAQDTAVYYCAR 1C1 952 RVTISVDNSKNQFSLKLSSLQAQDTAVYYCAR 1C1 953 RVTTSVDTSKSQVSFKMSSVTAADTAVYYCAR 1C1 954 RVTISVDTSKSQVSFKLSSLTAADTAVYYCAR 1C1 955 RVTISVDTSKSQVSFKLSSVQAADTAVYYCAR 1C1 956 RVTISVDTSKSQVSFKLSSVTAQDTAVYYCAR 1C1 957 RVTISVDTSKSQVSLKMSSLTAADTAVYYCAR 1C1 958 RVTISVDTSKSQVSLKMSSVQAADTAVYYCAR 1C1 959 RVTISVDTSKSQVSLKMSSVTAQDTAVYYCAR 1C1 960 RVTISVDTSKSQVSLKLSSLQAADTAVYYCAR 1C1 961 RVTISVDTSKSQVSLKLSSLTAQDTAVYYCAR 1C1 962 RVTISVDTSKSQVSLKLSSVQAQDTAVYYCAR 1C1 963 RVTISVDTSKSQFSFKMSSLTAADTAVYYCAR 1C1 964 RVTISVDTSKSQFSFKMSSVQAADTAVYYCAR 1C1 965 RVTISVDTSKSQFSFKMSSVTAQDTAVYYCAR 1C1 966 RVTISVDTSKSQFSFKLSSLQAADTAVYYCAR 1C1 967 RVTISVDTSKSQFSFKLSSLTAQDTAVYYCAR 1C1 968 RVTISVDTSKSQFSFKLSSVQAQDTAVYYCAR 1C1 969 RVTISVDTSKSQFSLKMSSLQAADTAVYYCAR 1C1 970 RVTISVDTSKSQFSLKMSSLTAQDTAVYYCAR 1C1 971 RVTISVDTSKSQFSLKMSSVQAQDTAVYYCAR 1C1 972 RVTISVDTSKSQFSLKLSSLQAQDTAVYYCAR 1C1 973 RVTISVDTSKNQVSFKMSSLTAADTAVYYCAR 1C1 974 RVTISVDTSKNQVSFKMSSVQAADTAVYYCAR 1C1 975 RVTISVDTSKNQVSFKMSSVTAQDTAVYYCAR 1C1 976 RVTISVDTSKNQVSFKLSSLQAADTAVYYCAR 1C1 977 RVTISVDTSKNQVSFKLSSLTAQDTAVYYCAR 1C1 978 RVTISVDTSKNQVSFKLSSVQAQDTAVYYCAR 1C1 979 RVTISVDTSKNQVSLKMSSLQAADTAVYYCAR 1C1 980 RVTISVDTSKNQVSLKMSSLTAQDTAVYYCAR 1C1 981 RVTISVDTSKNQVSLKMSSVQAQDTAVYYCAR 1C1 982 RVTTSVDTSKNQVSLKLSSLQAQDTAVYYCAR 1C1 983 RVTISVDTSKNQFSFKMSSLQAADTAVYYCAR 1C1 984 RVTISVDTSKNQFSFKMSSLTAQDTAVYYCAR 1C1 985 RVTISVDTSKNQFSFKMSSVQAQDTAVYYCAR 1C1 986 RVTISVDTSKNQFSFKLSSLQAQDTAVYYCAR 1C1 987 RVTISVDTSKNQFSLKMSSLQAQDTAVYYCAR 1C1 988 RLTISKDNSKSQVSLKLSSVTAADTAVYYCAR 1C1 989 RLTISKDNSKSQFSFKLSSVTAADTAVYYCAR 1C1 990 RLTISKDNSKSQFSLKMSSVTAADTAVYYCAR 1C1 991 RLTISKDNSKSQFSLKLSSLTAADTAVYYCAR 1C1 992 RLTISKDNSKSQFSLKLSSVQAADTAVYYCAR 1C1 993 RLTISKDNSKSQFSLKLSSVTAQDTAVYYCAR 1C1 994 RLTISKDNSKNQVSFKLSSVTAADTAVYYCAR 1C1 995 RLTISKDNSKNQVSLKMSSVTAADTAVYYCAR 1C1 996 RLTISKDNSKNQVSLKLSSLTAADTAVYYCAR 1C1 997 RLTISKDNSKNQVSLKLSSVQAADTAVYYCAR 1C1 998 RLTISKDNSKNQVSLKLSSVTAQDTAVYYCAR 1C1 999 RLTISKDNSKNQFSFKMSSVTAADTAVYYCAR 1C1 1000 RLTISKDNSKNQFSFKLSSLTAADTAVYYCAR 1C1 1001 RLTISKDNSKNQFSFKLSSVQAADTAVYYCAR 1C1 1002 RLTISKDNSKNQFSFKLSSVTAQDTAVYYCAR 1C1 1003 RLTISKDNSKNQFSLKMSSLTAADTAVYYCAR 1C1 1004 RLTISKDNSKNQFSLKMSSVQAADTAVYYCAR 1C1 1005 RLTISKDNSKNQFSLKMSSVTAQDTAVYYCAR 1C1 1006 RLTISKDNSKNQFSLKLSSLQAADTAVYYCAR 1C1 1007 RLTISKDNSKNQFSLKLSSLTAQDTAVYYCAR 1C1 1008 RLTISKDNSKNQFSLKLSSVQAQDTAVYYCAR 1C1 1009 RLTISKDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 1010 RLTISKDTSKSQVSLKMSSVTAADTAVYYCAR 1C1 1011 RLTTSKDTSKSQVSLKLSSLTAADTAVYYCAR 1C1 1012 RLTISKDTSKSQVSLKLSSVQAADTAVYYCAR 1C1 1013 RLTISKDTSKSQVSLKLSSVTAQDTAVYYCAR 1C1 1014 RLTISKDTSKSQFSFKMSSVTAADTAVYYCAR 1C1 1015 RLTISKDTSKSQFSFKLSSLTAADTAVYYCAR 1C1 1016 RLTISKDTSKSQFSFKLSSVQAADTAVYYCAR 1C1 1017 RLTISKDTSKSQFSFKLSSVTAQDTAVYYCAR 1C1 1018 RLTISKDTSKSQFSLKMSSLTAADTAVYYCAR 1C1 1019 RLTISKDTSKSQFSLKMSSVQAADTAVYYCAR 1C1 1020 RLTISKDTSKSQFSLKMSSVTAQDTAVYYCAR 1C1 1021 RLTISKDTSKSQFSLKLSSLQAADTAVYYCAR 1C1 1022 RLTISKDTSKSQFSLKLSSLTAQDTAVYYCAR 1C1 1023 RLTISKDTSKSQFSLKLSSVQAQDTAVYYCAR 1C1 1024 RLTISKDTSKNQVSFKMSSVTAADTAVYYCAR 1C1 1025 RLTISKDTSKNQVSFKLSSLTAADTAVYYCAR 1C1 1026 RLTISKDTSKNQVSFKLSSVQAADTAVYYCAR 1C1 1027 RLTISKDTSKNQVSFKLSSVTAQDTAVYYCAR 1C1 1028 RLTISKDTSKNQVSLKMSSLTAADTAVYYCAR 1C1 1029 RLTISKDTSKNQVSLKMSSVQAADTAVYYCAR 1C1 1030 RLTISKDTSKNQVSLKMSSVTAQDTAVYYCAR 1C1 1031 RLTISKDTSKNQVSLKLSSLQAADTAVYYCAR 1C1 1032 RLTISKDTSKNQVSLKLSSLTAQDTAVYYCAR 1C1 1033 RLTISKDTSKNQVSLKLSSVQAQDTAVYYCAR 1C1 1034 RLTISKDTSKNQFSFKMSSLTAADTAVYYCAR 1C1 1035 RLTISKDTSKNQFSFKMSSVQAADTAVYYCAR 1C1 1036 RLTISKDTSKNQFSFKMSSVTAQDTAVYYCAR 1C1 1037 RLTISKDTSKNQFSFKLSSLQAADTAVYYCAR 1C1 1038 RLTISKDTSKNQFSFKLSSLTAQDTAVYYCAR 1C1 1039 RLTISKDTSKNQFSFKLSSVQAQDTAVYYCAR 1C1 1040 RLTISKDTSKNQFSLKMSSLQAADTAVYYCAR 1C1 1041 RLTISKDTSKNQFSLKMSSLTAQDTAVYYCAR 1C1 1042 RLTISKDTSKNQFSLKMSSVQAQDTAVYYCAR 1C1 1043 RLTISKDTSKNQFSLKLSSLQAQDTAVYYCAR 1C1 1044 RLTISVDNSKSQVSFKLSSVTAADTAVYYCAR 1C1 1045 RLTISVDNSKSQVSLKMSSVTAADTAVYYCAR 1C1 1046 RLTISVDNSKSQVSLKLSSLTAADTAVYYCAR 1C1 1047 RLTISVDNSKSQVSLKLSSVQAADTAVYYCAR 1C1 1048 RLTISVDNSKSQVSLKLSSVTAQDTAVYYCAR 1C1 1049 RLTISVDNSKSQFSFKMSSVTAADTAVYYCAR 1C1 1050 RLTISVDNSKSQFSFKLSSLTAADTAVYYCAR 1C1 1051 RLTISVDNSKSQFSFKLSSVQAADTAVYYCAR 1C1 1052 RLTISVDNSKSQFSFKLSSVTAQDTAVYYCAR 1C1 1053 RLTISVDNSKSQFSLKMSSLTAADTAVYYCAR 1C1 1054 RLTISVDNSKSQFSLKMSSVQAADTAVYYCAR 1C1 1055 RLTISVDNSKSQFSLKMSSVTAQDTAVYYCAR 1C1 1056 RLTISVDNSKSQFSLKLSSLQAADTAVYYCAR 1C1 1057 RLTISVDNSKSQFSLKLSSLTAQDTAVYYCAR 1C1 1058 RLTISVDNSKSQFSLKLSSVQAQDTAVYYCAR 1C1 1059 RLTISVDNSKNQVSFKMSSVTAADTAVYYCAR 1C1 1060 RLTISVDNSKNQVSFKLSSLTAADTAVYYCAR 1C1 1061 RLTISVDNSKNQVSFKLSSVQAADTAVYYCAR 1C1 1062 RLTISVDNSKNQVSFKLSSVTAQDTAVYYCAR 1C1 1063 RLTISVDNSKNQVSLKMSSLTAADTAVYYCAR 1C1 1064 RLTISVDNSKNQVSLKMSSVQAADTAVYYCAR 1C1 1065 RLTISVDNSKNQVSLKMSSVTAQDTAVYYCAR 1C1 1066 RLTISVDNSKNQVSLKLSSLQAADTAVYYCAR 1C1 1067 RLTISVDNSKNQVSLKLSSLTAQDTAVYYCAR 1C1 1068 RLTISVDNSKNQVSLKLSSVQAQDTAVYYCAR 1C1 1069 RLTTSVDNSKNQFSFKMSSLTAADTAVYYCAR 1C1 1070 RLTISVDNSKNQFSFKMSSVQAADTAVYYCAR 1C1 1071 RLTISVDNSKNQFSFKMSSVTAQDTAVYYCAR 1C1 1072 RLTISVDNSKNQFSFKLSSLQAADTAVYYCAR 1C1 1073 RLTISVDNSKNQFSFKLSSLTAQDTAVYYCAR 1C1 1074 RLTISVDNSKNQFSFKLSSVQAQDTAVYYCAR 1C1 1075 RLTISVDNSKNQFSLKMSSLQAADTAVYYCAR 1C1 1076 RLTISVDNSKNQFSLKMSSLTAQDTAVYYCAR 1C1 1077 RLTISVDNSKNQFSLKMSSVQAQDTAVYYCAR 1C1 1078 RLTISVDNSKNQFSLKLSSLQAQDTAVYYCAR 1C1 1079 RLTISVDTSKSQVSFKMSSVTAADTAVYYCAR 1C1 1080 RLTISVDTSKSQVSFKLSSLTAADTAVYYCAR 1C1 1081 RLTISVDTSKSQVSFKLSSVQAADTAVYYCAR 1C1 1082 RLTISVDTSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1083 RLTISVDTSKSQVSLKMSSLTAADTAVYYCAR 1C1 1084 RLTISVDTSKSQVSLKMSSVQAADTAVYYCAR 1C1 1085 RLTISVDTSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1086 RLTISVDTSKSQVSLKLSSLQAADTAVYYCAR 1C1 1087 RLTISVDTSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1088 RLTISVDTSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1089 RLTISVDTSKSQFSFKMSSLTAADTAVYYCAR 1C1 1090 RLTISVDTSKSQFSFKMSSVQAADTAVYYCAR 1C1 1091 RLTISVDTSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1092 RLTISVDTSKSQFSFKLSSLQAADTAVYYCAR 1C1 1093 RLTISVDTSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1094 RLTISVDTSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1095 RLTISVDTSKSQFSLKMSSLQAADTAVYYCAR 1C1 1096 RLTISVDTSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1097 RLTISVDTSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1098 RLTISVDTSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1099 RLTISVDTSKNQVSFKMSSLTAADTAVYYCAR 1C1 1100 RLTISVDTSKNQVSFKMSSVQAADTAVYYCAR 1C1 1101 RLTISVDTSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1102 RLTISVDTSKNQVSFKLSSLQAADTAVYYCAR 1C1 1103 RLTISVDTSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1104 RLTISVDTSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1105 RLTISVDTSKNQVSLKMSSLQAADTAVYYCAR 1C1 1106 RLTISVDTSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1107 RLTISVDTSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1108 RLTISVDTSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1109 RLTISVDTSKNQFSFKMSSLQAADTAVYYCAR 1C1 1110 RLTISVDTSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1111 RLTISVDTSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1112 RLTISVDTSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1113 RLTISVDTSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1114 RVTISKDNSKSQVSFKLSSVTAADTAVYYCAR 1C1 1115 RVTISKDNSKSQVSLKMSSVTAADTAVYYCAR 1C1 1116 RVTISKDNSKSQVSLKLSSLTAADTAVYYCAR 1C1 1117 RVTISKDNSKSQVSLKLSSVQAADTAVYYCAR 1C1 1118 RVTISKDNSKSQVSLKLSSVTAQDTAVYYCAR 1C1 1119 RVTISKDNSKSQFSFKMSSVTAADTAVYYCAR 1C1 1120 RVTISKDNSKSQFSFKLSSLTAADTAVYYCAR 1C1 1121 RVTISKDNSKSQFSFKLSSVQAADTAVYYCAR 1C1 1122 RVTISKDNSKSQFSFKLSSVTAQDTAVYYCAR 1C1 1123 RVTISKDNSKSQFSLKMSSLTAADTAVYYCAR 1C1 1124 RVTISKDNSKSQFSLKMSSVQAADTAVYYCAR 1C1 1125 RVTISKDNSKSQFSLKMSSVTAQDTAVYYCAR 1C1 1126 RVTISKDNSKSQFSLKLSSLQAADTAVYYCAR 1C1 1127 RVTISKDNSKSQFSLKLSSLTAQDTAVYYCAR 1C1 1128 RVTISKDNSKSQFSLKLSSVQAQDTAVYYCAR 1C1 1129 RVTISKDNSKNQVSFKMSSVTAADTAVYYCAR 1C1 1130 RVTISKDNSKNQVSFKLSSLTAADTAVYYCAR 1C1 1131 RVTISKDNSKNQVSFKLSSVQAADTAVYYCAR 1C1 1132 RVTISKDNSKNQVSFKLSSVTAQDTAVYYCAR 1C1 1133 RVTISKDNSKNQVSLKMSSLTAADTAVYYCAR 1C1 1134 RVTISKDNSKNQVSLKMSSVQAADTAVYYCAR 1C1 1135 RVTISKDNSKNQVSLKMSSVTAQDTAVYYCAR 1C1 1136 RVTISKDNSKNQVSLKLSSLQAADTAVYYCAR 1C1 1137 RVTISKDNSKNQVSLKLSSLTAQDTAVYYCAR 1C1 1138 RVTISKDNSKNQVSLKLSSVQAQDTAVYYCAR 1C1 1139 RVTISKDNSKNQFSFKMSSLTAADTAVYYCAR 1C1 1140 RVTISKDNSKNQFSFKMSSVQAADTAVYYCAR 1C1 1141 RVTISKDNSKNQFSFKMSSVTAQDTAVYYCAR 1C1 1142 RVTISKDNSKNQFSFKLSSLQAADTAVYYCAR 1C1 1143 RVTISKDNSKNQFSFKLSSLTAQDTAVYYCAR 1C1 1144 RVTISKDNSKNQFSFKLSSVQAQDTAVYYCAR 1C1 1145 RVTISKDNSKNQFSLKMSSLQAADTAVYYCAR 1C1 1146 RVTISKDNSKNQFSLKMSSLTAQDTAVYYCAR 1C1 1147 RVTISKDNSKNQFSLKMSSVQAQDTAVYYCAR 1C1 1148 RVTISKDNSKNQFSLKLSSLQAQDTAVYYCAR 1C1 1149 RVTISKDTSKSQVSFKMSSVTAADTAVYYCAR 1C1 1150 RVTISKDTSKSQVSFKLSSLTAADTAVYYCAR 1C1 1151 RVTISKDTSKSQVSFKLSSVQAADTAVYYCAR 1C1 1152 RVTISKDTSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1153 RVTISKDTSKSQVSLKMSSLTAADTAVYYCAR 1C1 1154 RVTISKDTSKSQVSLKMSSVQAADTAVYYCAR 1C1 1155 RVTISKDTSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1156 RVTISKDTSKSQVSLKLSSLQAADTAVYYCAR 1C1 1157 RVTISKDTSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1158 RVTISKDTSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1159 RVTISKDTSKSQFSFKMSSLTAADTAVYYCAR 1C1 1160 RVTISKDTSKSQFSFKMSSVQAADTAVYYCAR 1C1 1161 RVTISKDTSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1162 RVTISKDTSKSQFSFKLSSLQAADTAVYYCAR 1C1 1163 RVTISKDTSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1164 RVTISKDTSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1165 RVTISKDTSKSQFSLKMSSLQAADTAVYYCAR 1C1 1166 RVTISKDTSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1167 RVTISKDTSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1168 RVTISKDTSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1169 RVTISKDTSKNQVSFKMSSLTAADTAVYYCAR 1C1 1170 RVTISKDTSKNQVSFKMSSVQAADTAVYYCAR 1C1 1171 RVTISKDTSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1172 RVTISKDTSKNQVSFKLSSLQAADTAVYYCAR 1C1 1173 RVTISKDTSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1174 RVTISKDTSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1175 RVTISKDTSKNQVSLKMSSLQAADTAVYYCAR 1C1 1176 RVTISKDTSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1177 RVTISKDTSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1178 RVTISKDTSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1179 RVTISKDTSKNQFSFKMSSLQAADTAVYYCAR 1C1 1180 RVTISKDTSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1181 RVTISKDTSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1182 RVTISKDTSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1183 RVTISKDTSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1184 RVTISVDNSKSQVSFKMSSVTAADTAVYYCAR 1C1 1185 RVTTSVDNSKSQVSFKLSSLTAADTAVYYCAR 1C1 1186 RVTISVDNSKSQVSFKLSSVQAADTAVYYCAR 1C1 1187 RVTISVDNSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1188 RVTISVDNSKSQVSLKMSSLTAADTAVYYCAR 1C1 1189 RVTISVDNSKSQVSLKMSSVQAADTAVYYCAR 1C1 1190 RVTISVDNSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1191 RVTISVDNSKSQVSLKLSSLQAADTAVYYCAR 1C1 1192 RVTISVDNSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1193 RVTISVDNSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1194 RVTISVDNSKSQFSFKMSSLTAADTAVYYCAR 1C1 1195 RVTISVDNSKSQFSFKMSSVQAADTAVYYCAR 1C1 1196 RVTISVDNSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1197 RVTISVDNSKSQFSFKLSSLQAADTAVYYCAR 1C1 1198 RVTISVDNSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1199 RVTISVDNSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1200 RVTISVDNSKSQFSLKMSSLQAADTAVYYCAR 1C1 1201 RVTISVDNSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1202 RVTISVDNSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1203 RVTISVDNSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1204 RVTISVDNSKNQVSFKMSSLTAADTAVYYCAR 1C1 1205 RVTISVDNSKNQVSFKMSSVQAADTAVYYCAR 1C1 1206 RVTISVDNSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1207 RVTISVDNSKNQVSFKLSSLQAADTAVYYCAR 1C1 1208 RVTISVDNSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1209 RVTISVDNSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1210 RVTISVDNSKNQVSLKMSSLQAADTAVYYCAR 1C1 1211 RVTISVDNSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1212 RVTISVDNSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1213 RVTISVDNSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1214 RVTTSVDNSKNQFSFKMSSLQAADTAVYYCAR 1C1 1215 RVTISVDNSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1216 RVTISVDNSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1217 RVTISVDNSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1218 RVTISVDNSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1219 RVTISVDTSKSQVSFKMSSLTAADTAVYYCAR 1C1 1220 RVTISVDTSKSQVSFKMSSVQAADTAVYYCAR 1C1 1221 RVTISVDTSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1222 RVTISVDTSKSQVSFKLSSLQAADTAVYYCAR 1C1 1223 RVTISVDTSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1224 RVTISVDTSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1225 RVTISVDTSKSQVSLKMSSLQAADTAVYYCAR 1C1 1226 RVTISVDTSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1227 RVTISVDTSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1228 RVTISVDTSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1229 RVTISVDTSKSQFSFKMSSLQAADTAVYYCAR 1C1 1230 RVTISVDTSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1231 RVTISVDTSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1232 RVTISVDTSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1233 RVTISVDTSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1234 RVTISVDTSKNQVSFKMSSLQAADTAVYYCAR 1C1 1235 RVTISVDTSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1236 RVTISVDTSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1237 RVTISVDTSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1238 RVTISVDTSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1239 RVTISVDTSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1240 RLTISKDNSKSQVSFKLSSVTAADTAVYYCAR 1C1 1241 RLTISKDNSKSQVSLKMSSVTAADTAVYYCAR 1C1 1242 RLTISKDNSKSQVSLKLSSLTAADTAVYYCAR 1C1 1243 RLTISKDNSKSQVSLKLSSVQAADTAVYYCAR 1C1 1244 RLTISKDNSKSQVSLKLSSVTAQDTAVYYCAR 1C1 1245 RLTISKDNSKSQFSFKMSSVTAADTAVYYCAR 1C1 1246 RLTISKDNSKSQFSFKLSSLTAADTAVYYCAR 1C1 1247 RLTISKDNSKSQFSFKLSSVQAADTAVYYCAR 1C1 1248 RLTISKDNSKSQFSFKLSSVTAQDTAVYYCAR 1C1 1249 RLTISKDNSKSQFSLKMSSLTAADTAVYYCAR 1C1 1250 RLTISKDNSKSQFSLKMSSVQAADTAVYYCAR 1C1 1251 RLTISKDNSKSQFSLKMSSVTAQDTAVYYCAR 1C1 1252 RLTISKDNSKSQFSLKLSSLQAADTAVYYCAR 1C1 1253 RLTISKDNSKSQFSLKLSSLTAQDTAVYYCAR 1C1 1254 RLTISKDNSKSQFSLKLSSVQAQDTAVYYCAR 1C1 1255 RLTISKDNSKNQVSFKMSSVTAADTAVYYCAR 1C1 1256 RLTISKDNSKNQVSFKLSSLTAADTAVYYCAR 1C1 1257 RLTISKDNSKNQVSFKLSSVQAADTAVYYCAR 1C1 1258 RLTISKDNSKNQVSFKLSSVTAQDTAVYYCAR 1C1 1259 RLTISKDNSKNQVSLKMSSLTAADTAVYYCAR 1C1 1260 RLTISKDNSKNQVSLKMSSVQAADTAVYYCAR 1C1 1261 RLTISKDNSKNQVSLKMSSVTAQDTAVYYCAR 1C1 1262 RLTISKDNSKNQVSLKLSSLQAADTAVYYCAR 1C1 1263 RLTISKDNSKNQVSLKLSSLTAQDTAVYYCAR 1C1 1264 RLTISKDNSKNQVSLKLSSVQAQDTAVYYCAR 1C1 1265 RLTISKDNSKNQFSFKMSSLTAADTAVYYCAR 1C1 1266 RLTISKDNSKNQFSFKMSSVQAADTAVYYCAR 1C1 1267 RLTISKDNSKNQFSFKMSSVTAQDTAVYYCAR 1C1 1268 RLTISKDNSKNQFSFKLSSLQAADTAVYYCAR 1C1 1269 RLTISKDNSKNQFSFKLSSLTAQDTAVYYCAR 1C1 1270 RLTISKDNSKNQFSFKLSSVQAQDTAVYYCAR 1C1 1271 RLTISKDNSKNQFSLKMSSLQAADTAVYYCAR 1C1 1272 RLTTSKDNSKNQFSLKMSSLTAQDTAVYYCAR 1C1 1273 RLTISKDNSKNQFSLKMSSVQAQDTAVYYCAR 1C1 1274 RLTISKDNSKNQFSLKLSSLQAQDTAVYYCAR 1C1 1275 RLTISKDTSKSQVSFKMSSVTAADTAVYYCAR 1C1 1276 RLTISKDTSKSQVSFKLSSLTAADTAVYYCAR 1C1 1277 RLTISKDTSKSQVSFKLSSVQAADTAVYYCAR 1C1 1278 RLTISKDTSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1279 RLTISKDTSKSQVSLKMSSLTAADTAVYYCAR 1C1 1280 RLTISKDTSKSQVSLKMSSVQAADTAVYYCAR 1C1 1281 RLTISKDTSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1282 RLTISKDTSKSQVSLKLSSLQAADTAVYYCAR 1C1 1283 RLTISKDTSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1284 RLTISKDTSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1285 RLTISKDTSKSQFSFKMSSLTAADTAVYYCAR 1C1 1286 RLTISKDTSKSQFSFKMSSVQAADTAVYYCAR 1C1 1287 RLTISKDTSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1288 RLTISKDTSKSQFSFKLSSLQAADTAVYYCAR 1C1 1289 RLTISKDTSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1290 RLTISKDTSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1291 RLTISKDTSKSQFSLKMSSLQAADTAVYYCAR 1C1 1292 RLTISKDTSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1293 RLTISKDTSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1294 RLTISKDTSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1295 RLTISKDTSKNQVSFKMSSLTAADTAVYYCAR 1C1 1296 RLTISKDTSKNQVSFKMSSVQAADTAVYYCAR 1C1 1297 RLTISKDTSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1298 RLTISKDTSKNQVSFKLSSLQAADTAVYYCAR 1C1 1299 RLTISKDTSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1300 RLTISKDTSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1301 RLTTSKDTSKNQVSLKMSSLQAADTAVYYCAR 1C1 1302 RLTISKDTSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1303 RLTISKDTSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1304 RLTISKDTSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1305 RLTISKDTSKNQFSFKMSSLQAADTAVYYCAR 1C1 1306 RLTISKDTSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1307 RLTISKDTSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1308 RLTISKDTSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1309 RLTISKDTSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1310 RLTISVDNSKSQVSFKMSSVTAADTAVYYCAR 1C1 1311 RLTISVDNSKSQVSFKLSSLTAADTAVYYCAR 1C1 1312 RLTISVDNSKSQVSFKLSSVQAADTAVYYCAR 1C1 1313 RLTISVDNSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1314 RLTISVDNSKSQVSLKMSSLTAADTAVYYCAR 1C1 1315 RLTISVDNSKSQVSLKMSSVQAADTAVYYCAR 1C1 1316 RLTISVDNSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1317 RLTISVDNSKSQVSLKLSSLQAADTAVYYCAR 1C1 1318 RLTISVDNSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1319 RLTISVDNSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1320 RLTISVDNSKSQFSFKMSSLTAADTAVYYCAR 1C1 1321 RLTISVDNSKSQFSFKMSSVQAADTAVYYCAR 1C1 1322 RLTISVDNSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1323 RLTISVDNSKSQFSFKLSSLQAADTAVYYCAR 1C1 1324 RLTISVDNSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1325 RLTISVDNSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1326 RLTISVDNSKSQFSLKMSSLQAADTAVYYCAR 1C1 1327 RLTISVDNSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1328 RLTISVDNSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1329 RLTISVDNSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1330 RLTTSVDNSKNQVSFKMSSLTAADTAVYYCAR 1C1 1331 RLTISVDNSKNQVSFKMSSVQAADTAVYYCAR 1C1 1332 RLTISVDNSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1333 RLTISVDNSKNQVSFKLSSLQAADTAVYYCAR 1C1 1334 RLTISVDNSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1335 RLTISVDNSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1336 RLTISVDNSKNQVSLKMSSLQAADTAVYYCAR 1C1 1337 RLTISVDNSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1338 RLTISVDNSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1339 RLTISVDNSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1340 RLTISVDNSKNQFSFKMSSLQAADTAVYYCAR 1C1 1341 RLTISVDNSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1342 RLTISVDNSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1343 RLTISVDNSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1344 RLTISVDNSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1345 RLTISVDTSKSQVSFKMSSLTAADTAVYYCAR 1C1 1346 RLTISVDTSKSQVSFKMSSVQAADTAVYYCAR 1C1 1347 RLTISVDTSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1348 RLTISVDTSKSQVSFKLSSLQAADTAVYYCAR 1C1 1349 RLTISVDTSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1350 RLTISVDTSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1351 RLTISVDTSKSQVSLKMSSLQAADTAVYYCAR 1C1 1352 RLTISVDTSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1353 RLTISVDTSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1354 RLTISVDTSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1355 RLTISVDTSKSQFSFKMSSLQAADTAVYYCAR 1C1 1356 RLTISVDTSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1357 RLTISVDTSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1358 RLTISVDTSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1359 RLTISVDTSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1360 RLTISVDTSKNQVSFKMSSLQAADTAVYYCAR 1C1 1361 RLTISVDTSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1362 RLTISVDTSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1363 RLTISVDTSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1364 RLTISVDTSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1365 RLTISVDTSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1366 RVTISKDNSKSQVSFKMSSVTAADTAVYYCAR 1C1 1367 RVTISKDNSKSQVSFKLSSLTAADTAVYYCAR 1C1 1368 RVTISKDNSKSQVSFKLSSVQAADTAVYYCAR 1C1 1369 RVTISKDNSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1370 RVTISKDNSKSQVSLKMSSLTAADTAVYYCAR 1C1 1371 RVTISKDNSKSQVSLKMSSVQAADTAVYYCAR 1C1 1372 RVTISKDNSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1373 RVTISKDNSKSQVSLKLSSLQAADTAVYYCAR 1C1 1374 RVTISKDNSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1375 RVTISKDNSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1376 RVTISKDNSKSQFSFKMSSLTAADTAVYYCAR 1C1 1377 RVTISKDNSKSQFSFKMSSVQAADTAVYYCAR 1C1 1378 RVTISKDNSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1379 RVTISKDNSKSQFSFKLSSLQAADTAVYYCAR 1C1 1380 RVTISKDNSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1381 RVTISKDNSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1382 RVTISKDNSKSQFSLKMSSLQAADTAVYYCAR 1C1 1383 RVTISKDNSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1384 RVTISKDNSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1385 RVTISKDNSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1386 RVTISKDNSKNQVSFKMSSLTAADTAVYYCAR 1C1 1387 RVTISKDNSKNQVSFKMSSVQAADTAVYYCAR 1C1 1388 RVTISKDNSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1389 RVTISKDNSKNQVSFKLSSLQAADTAVYYCAR 1C1 1390 RVTISKDNSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1391 RVTISKDNSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1392 RVTISKDNSKNQVSLKMSSLQAADTAVYYCAR 1C1 1393 RVTISKDNSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1394 RVTISKDNSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1395 RVTISKDNSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1396 RVTISKDNSKNQFSFKMSSLQAADTAVYYCAR 1C1 1397 RVTISKDNSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1398 RVTISKDNSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1399 RVTISKDNSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1400 RVTISKDNSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1401 RVTISKDTSKSQVSFKMSSLTAADTAVYYCAR 1C1 1402 RVTISKDTSKSQVSFKMSSVQAADTAVYYCAR 1C1 1403 RVTISKDTSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1404 RVTISKDTSKSQVSFKLSSLQAADTAVYYCAR 1C1 1405 RVTISKDTSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1406 RVTISKDTSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1407 RVTISKDTSKSQVSLKMSSLQAADTAVYYCAR 1C1 1408 RVTISKDTSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1409 RVTISKDTSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1410 RVTISKDTSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1411 RVTISKDTSKSQFSFKMSSLQAADTAVYYCAR 1C1 1412 RVTISKDTSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1413 RVTISKDTSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1414 RVTISKDTSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1415 RVTISKDTSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1416 RVTISKDTSKNQVSFKMSSLQAADTAVYYCAR 1C1 1417 RVTISKDTSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1418 RVTISKDTSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1419 RVTISKDTSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1420 RVTISKDTSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1421 RVTISKDTSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1422 RVTISVDNSKSQVSFKMSSLTAADTAVYYCAR 1C1 1423 RVTISVDNSKSQVSFKMSSVQAADTAVYYCAR 1C1 1424 RVTISVDNSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1425 RVTISVDNSKSQVSFKLSSLQAADTAVYYCAR 1C1 1426 RVTISVDNSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1427 RVTISVDNSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1428 RVTISVDNSKSQVSLKMSSLQAADTAVYYCAR 1C1 1429 RVTISVDNSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1430 RVTISVDNSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1431 RVTISVDNSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1432 RVTISVDNSKSQFSFKMSSLQAADTAVYYCAR 1C1 1433 RVTISVDNSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1434 RVTISVDNSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1435 RVTISVDNSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1436 RVTISVDNSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1437 RVTISVDNSKNQVSFKMSSLQAADTAVYYCAR 1C1 1438 RVTISVDNSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1439 RVTISVDNSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1440 RVTISVDNSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1441 RVTISVDNSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1442 RVTISVDNSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1443 RVTISVDTSKSQVSFKMSSLQAADTAVYYCAR 1C1 1444 RVTISVDTSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1445 RVTISVDTSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1446 RVTISVDTSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1447 RVTISVDTSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1448 RVTISVDTSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1449 RVTISVDTSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1450 RLTISKDNSKSQVSFKMSSVTAADTAVYYCAR 1C1 1451 RLTISKDNSKSQVSFKLSSLTAADTAVYYCAR 1C1 1452 RLTISKDNSKSQVSFKLSSVQAADTAVYYCAR 1C1 1453 RLTISKDNSKSQVSFKLSSVTAQDTAVYYCAR 1C1 1454 RLTISKDNSKSQVSLKMSSLTAADTAVYYCAR 1C1 1455 RLTISKDNSKSQVSLKMSSVQAADTAVYYCAR 1C1 1456 RLTISKDNSKSQVSLKMSSVTAQDTAVYYCAR 1C1 1457 RLTISKDNSKSQVSLKLSSLQAADTAVYYCAR 1C1 1458 RLTISKDNSKSQVSLKLSSLTAQDTAVYYCAR 1C1 1459 RLTISKDNSKSQVSLKLSSVQAQDTAVYYCAR 1C1 1460 RLTISKDNSKSQFSFKMSSLTAADTAVYYCAR 1C1 1461 RLTISKDNSKSQFSFKMSSVQAADTAVYYCAR 1C1 1462 RLTISKDNSKSQFSFKMSSVTAQDTAVYYCAR 1C1 1463 RLTISKDNSKSQFSFKLSSLQAADTAVYYCAR 1C1 1464 RLTISKDNSKSQFSFKLSSLTAQDTAVYYCAR 1C1 1465 RLTISKDNSKSQFSFKLSSVQAQDTAVYYCAR 1C1 1466 RLTISKDNSKSQFSLKMSSLQAADTAVYYCAR 1C1 1467 RLTISKDNSKSQFSLKMSSLTAQDTAVYYCAR 1C1 1468 RLTISKDNSKSQFSLKMSSVQAQDTAVYYCAR 1C1 1469 RLTISKDNSKSQFSLKLSSLQAQDTAVYYCAR 1C1 1470 RLTISKDNSKNQVSFKMSSLTAADTAVYYCAR 1C1 1471 RLTISKDNSKNQVSFKMSSVQAADTAVYYCAR 1C1 1472 RLTISKDNSKNQVSFKMSSVTAQDTAVYYCAR 1C1 1473 RLTISKDNSKNQVSFKLSSLQAADTAVYYCAR 1C1 1474 RLTISKDNSKNQVSFKLSSLTAQDTAVYYCAR 1C1 1475 RLTISKDNSKNQVSFKLSSVQAQDTAVYYCAR 1C1 1476 RLTISKDNSKNQVSLKMSSLQAADTAVYYCAR 1C1 1477 RLTISKDNSKNQVSLKMSSLTAQDTAVYYCAR 1C1 1478 RLTISKDNSKNQVSLKMSSVQAQDTAVYYCAR 1C1 1479 RLTISKDNSKNQVSLKLSSLQAQDTAVYYCAR 1C1 1480 RLTISKDNSKNQFSFKMSSLQAADTAVYYCAR 1C1 1481 RLTISKDNSKNQFSFKMSSLTAQDTAVYYCAR 1C1 1482 RLTISKDNSKNQFSFKMSSVQAQDTAVYYCAR 1C1 1483 RLTISKDNSKNQFSFKLSSLQAQDTAVYYCAR 1C1 1484 RLTISKDNSKNQFSLKMSSLQAQDTAVYYCAR 1C1 1485 RLTISKDTSKSQVSFKMSSLTAADTAVYYCAR 1C1 1486 RLTISKDTSKSQVSFKMSSVQAADTAVYYCAR 1C1 1487 RLTISKDTSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1488 RLTISKDTSKSQVSFKLSSLQAADTAVYYCAR 1C1 1489 RLTISKDTSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1490 RLTISKDTSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1491 RLTISKDTSKSQVSLKMSSLQAADTAVYYCAR 1C1 1492 RLTISKDTSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1493 RLTISKDTSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1494 RLTISKDTSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1495 RLTISKDTSKSQFSFKMSSLQAADTAVYYCAR 1C1 1496 RLTISKDTSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1497 RLTISKDTSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1498 RLTISKDTSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1499 RLTISKDTSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1500 RLTISKDTSKNQVSFKMSSLQAADTAVYYCAR 1C1 1501 RLTISKDTSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1502 RLTISKDTSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1503 RLTISKDTSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1504 RLTISKDTSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1505 RLTISKDTSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1506 RLTISVDNSKSQVSFKMSSLTAADTAVYYCAR 1C1 1507 RLTISVDNSKSQVSFKMSSVQAADTAVYYCAR 1C1 1508 RLTISVDNSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1509 RLTISVDNSKSQVSFKLSSLQAADTAVYYCAR 1C1 1510 RLTISVDNSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1511 RLTISVDNSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1512 RLTISVDNSKSQVSLKMSSLQAADTAVYYCAR 1C1 1513 RLTISVDNSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1514 RLTISVDNSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1515 RLTISVDNSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1516 RLTISVDNSKSQFSFKMSSLQAADTAVYYCAR 1C1 1517 RLTISVDNSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1518 RLTISVDNSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1519 RLTISVDNSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1520 RLTISVDNSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1521 RLTISVDNSKNQVSFKMSSLQAADTAVYYCAR 1C1 1522 RLTISVDNSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1523 RLTISVDNSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1524 RLTISVDNSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1525 RLTISVDNSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1526 RLTISVDNSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1527 RLTISVDTSKSQVSFKMSSLQAADTAVYYCAR 1C1 1528 RLTISVDTSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1529 RLTISVDTSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1530 RLTISVDTSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1531 RLTISVDTSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1532 RLTISVDTSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1533 RLTISVDTSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1534 RVTISKDNSKSQVSFKMSSLTAADTAVYYCAR 1C1 1535 RVTISKDNSKSQVSFKMSSVQAADTAVYYCAR 1C1 1536 RVTISKDNSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1537 RVTISKDNSKSQVSFKLSSLQAADTAVYYCAR 1C1 1538 RVTISKDNSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1539 RVTISKDNSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1540 RVTISKDNSKSQVSLKMSSLQAADTAVYYCAR 1C1 1541 RVTISKDNSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1542 RVTISKDNSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1543 RVTISKDNSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1544 RVTISKDNSKSQFSFKMSSLQAADTAVYYCAR 1C1 1545 RVTISKDNSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1546 RVTISKDNSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1547 RVTISKDNSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1548 RVTISKDNSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1549 RVTISKDNSKNQVSFKMSSLQAADTAVYYCAR 1C1 1550 RVTISKDNSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1551 RVTISKDNSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1552 RVTISKDNSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1553 RVTISKDNSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1554 RVTISKDNSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1555 RVTISKDTSKSQVSFKMSSLQAADTAVYYCAR 1C1 1556 RVTISKDTSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1557 RVTISKDTSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1558 RVTISKDTSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1559 RVTISKDTSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1560 RVTISKDTSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1561 RVTISKDTSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1562 RVTISVDNSKSQVSFKMSSLQAADTAVYYCAR 1C1 1563 RVTISVDNSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1564 RVTISVDNSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1565 RVTISVDNSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1566 RVTISVDNSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1567 RVTISVDNSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1568 RVTISVDNSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1569 RVTISVDTSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1570 RLTISKDNSKSQVSFKMSSLTAADTAVYYCAR 1C1 1571 RLTISKDNSKSQVSFKMSSVQAADTAVYYCAR 1C1 1572 RLTISKDNSKSQVSFKMSSVTAQDTAVYYCAR 1C1 1573 RLTISKDNSKSQVSFKLSSLQAADTAVYYCAR 1C1 1574 RLTISKDNSKSQVSFKLSSLTAQDTAVYYCAR 1C1 1575 RLTISKDNSKSQVSFKLSSVQAQDTAVYYCAR 1C1 1576 RLTISKDNSKSQVSLKMSSLQAADTAVYYCAR 1C1 1577 RLTISKDNSKSQVSLKMSSLTAQDTAVYYCAR 1C1 1578 RLTISKDNSKSQVSLKMSSVQAQDTAVYYCAR 1C1 1579 RLTISKDNSKSQVSLKLSSLQAQDTAVYYCAR 1C1 1580 RLTISKDNSKSQFSFKMSSLQAADTAVYYCAR 1C1 1581 RLTISKDNSKSQFSFKMSSLTAQDTAVYYCAR 1C1 1582 RLTISKDNSKSQFSFKMSSVQAQDTAVYYCAR 1C1 1583 RLTISKDNSKSQFSFKLSSLQAQDTAVYYCAR 1C1 1584 RLTISKDNSKSQFSLKMSSLQAQDTAVYYCAR 1C1 1585 RLTISKDNSKNQVSFKMSSLQAADTAVYYCAR 1C1 1586 RLTISKDNSKNQVSFKMSSLTAQDTAVYYCAR 1C1 1587 RLTISKDNSKNQVSFKMSSVQAQDTAVYYCAR 1C1 1588 RLTISKDNSKNQVSFKLSSLQAQDTAVYYCAR 1C1 1589 RLTISKDNSKNQVSLKMSSLQAQDTAVYYCAR 1C1 1590 RLTISKDNSKNQFSFKMSSLQAQDTAVYYCAR 1C1 1591 RLTISKDTSKSQVSFKMSSLQAADTAVYYCAR 1C1 1592 RLTISKDTSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1593 RLTISKDTSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1594 RLTISKDTSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1595 RLTISKDTSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1596 RLTISKDTSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1597 RLTISKDTSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1598 RLTISVDNSKSQVSFKMSSLQAADTAVYYCAR 1C1 1599 RLTISVDNSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1600 RLTISVDNSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1601 RLTISVDNSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1602 RLTISVDNSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1603 RLTISVDNSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1604 RLTISVDNSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1605 RLTISVDTSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1606 RVTISKDNSKSQVSFKMSSLQAADTAVYYCAR 1C1 1607 RVTISKDNSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1608 RVTISKDNSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1609 RVTISKDNSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1610 RVTISKDNSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1611 RVTISKDNSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1612 RVTISKDNSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1613 RVTISKDTSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1614 RVTISVDNSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1615 RLTISKDNSKSQVSFKMSSLQAADTAVYYCAR 1C1 1616 RLTISKDNSKSQVSFKMSSLTAQDTAVYYCAR 1C1 1617 RLTISKDNSKSQVSFKMSSVQAQDTAVYYCAR 1C1 1618 RLTISKDNSKSQVSFKLSSLQAQDTAVYYCAR 1C1 1619 RLTISKDNSKSQVSLKMSSLQAQDTAVYYCAR 1C1 1620 RLTISKDNSKSQFSFKMSSLQAQDTAVYYCAR 1C1 1621 RLTISKDNSKNQVSFKMSSLQAQDTAVYYCAR 1C1 1622 RLTISKDTSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1623 RLTISVDNSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1624 RVTISKDNSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1625 RLTISKDNSKSQVSFKMSSLQAQDTAVYYCAR 1C1 1626 RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 1627 RLTISRDTSKNQFSLKLSSVTAADTAVYYCAR 1C1 1628 RVTISRDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 1629 RVTISRDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 1630 RVTISRDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 1631 RLTISRDTSKSQFSLKLSSVTAADTAVYYCAR 1C1 1632 RLTISRDTSKNQVSLKLSSVTAADTAVYYCAR 1C1 1633 RLTISRDTSKNQFSFKLSSVTAADTAVYYCAR 1C1 1634 RVTISRDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 1635 RVTISRDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 1636 RVTISRDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 1637 RLTISRDTSKSQVSLKLSSVTAADTAVYYCAR 1C1 1638 RLTISRDTSKSQFSFKLSSVTAADTAVYYCAR 1C1 1639 RLTISRDTSKNQVSFKLSSVTAADTAVYYCAR 1C1 1640 RVTISRDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 1641 RLTISRDTSKSQVSFKLSSVTAADTAVYYCAR 1C1 1642 RVTITADESTSTAYMELSSLRSEDTAVYYCAR 3P10, 5F12, 25M22, 1643 17J16 RFTITADESTSTAYMELSSLRSEDTAVYYCAR 3P10 1644 RVTFTADESTSTAYMELSSLRSEDTAVYYCAR 3P10 1645 RVTITLDESTSTAYMELSSLRSEDTAVYYCAR 3P10 1646 RVTITADESTSTAYMELSNLRSEDTAVYYCAR 3P10 1647 RVTITADESTSTAYMELSSLRSEDTAVFYCAR 3P10 1648 RFTFTADESTSTAYMELSSLRSEDTAVYYCAR 3P10 1649 RFTITLDESTSTAYMELSSLRSEDTAVYYCAR 3P10 1650 RFTITADESTSTAYMELSNLRSEDTAVYYCAR 3P10 1651 RFTITADESTSTAYMELSSLRSEDTAVFYCAR 3P10 1652 RVTFTLDESTSTAYMELSSLRSEDTAVYYCAR 3P10 1653 RVTFTADESTSTAYMELSNLRSEDTAVYYCAR 3P10 1654 RVTFTADESTSTAYMELSSLRSEDTAVFYCAR 3P10 1655 RVTITLDESTSTAYMELSNLRSEDTAVYYCAR 3P10 1656 RVTITLDESTSTAYMELSSLRSEDTAVFYCAR 3P10 1657 RVTITADESTSTAYMELSNLRSEDTAVFYCAR 3P10 1658 RFTFTLDESTSTAYMELSSLRSEDTAVYYCAR 3P10 1659 RFTFTADESTSTAYMELSNLRSEDTAVYYCAR 3P10 1660 RFTFTADESTSTAYMELSSLRSEDTAVFYCAR 3P10 1661 RVTFTLDESTSTAYMELSNLRSEDTAVYYCAR 3P10 1662 RVTFTLDESTSTAYMELSSLRSEDTAVFYCAR 3P10 1663 RVTFTADESTSTAYMELSNLRSEDTAVFYCAR 3P10 1664 RVTITLDESTSTAYMELSNLRSEDTAVFYCAR 3P10 1665 RFTFTLDESTSTAYMELSNLRSEDTAVYYCAR 3P10 1666 RFTFTLDESTSTAYMELSSLRSEDTAVFYCAR 3P10 1667 RFTFTADESTSTAYMELSNLRSEDTAVFYCAR 3P10 1668 RVTFTLDESTSTAYMELSNLRSEDTAVFYCAR 3P10 1669 RFTFTLDESTSTAYMELSNLRSEDTAVFYCAR 3P10 1670 RVTLTADTSTDTAYMELSSLRSEDTAVYFCAR 3P10 1671 RFTLTADTSTDTAYMELSSLRSEDTAVYFCAR 3P10 1672 RVTFTADTSTDTAYMELSSLRSEDTAVYFCAR 3P10 1673 RVTLTADTSTDTAYLELSSLRSEDTAVYFCAR 3P10 1674 RFTFTADTSTDTAYMELSSLRSEDTAVYFCAR 3P10 1675 RFTLTADTSTDTAYLELSSLRSEDTAVYFCAR 3P10 1676 RVTFTADTSTDTAYLELSSLRSEDTAVYFCAR 3P10 1677 RFTFTADTSTDTAYLELSSLRSEDTAVYFCAR 3P10 1678 RATITADESTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22, 17J16 1679 RVTLTADESTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22, 17J16 1680 RVTITADKSTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22 1681 RATLTADESTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22, 17J16 1682 RATITADKSTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22 1683 RVTLTADKSTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22 1684 RATLTADKSTSTAYMELSSLRSEDTAVYYCAR 5F12, 25M22 1685 RATITADESTSTAYMELSSLRSEDTAVYYCAR 17J16 1686 RVTITADESSSTAYMELSSLRSEDTAVYYCAR 17J16 1687 RVTITADESTSTAYLELSSLRSEDTAVYYCAR 17J16 1688 RVTITADESTSTAYMELSRLRSEDTAVYYCAR 17J16 1689 RATITADESSSTAYMELSSLRSEDTAVYYCAR 17J16 1690 RATITADESTSTAYLELSSLRSEDTAVYYCAR 17J16 1691 RATITADESTSTAYMELSRLRSEDTAVYYCAR 17J16 1692 RVTLTADESSSTAYMELSSLRSEDTAVYYCAR 17J16 1693 RVTLTADESTSTAYLELSSLRSEDTAVYYCAR 17J16 1694 RVTLTADESTSTAYMELSRLRSEDTAVYYCAR 17J16 1695 RVTITADESSSTAYLELSSLRSEDTAVYYCAR 17J16 1696 RVTITADESSSTAYMELSRLRSEDTAVYYCAR 17J16 1697 RVTITADESTSTAYLELSRLRSEDTAVYYCAR 17J16 1698 RATLTADESSSTAYMELSSLRSEDTAVYYCAR 17J16 1699 RATLTADESTSTAYLELSSLRSEDTAVYYCAR 17J16 1700 RATLTADESTSTAYMELSRLRSEDTAVYYCAR 17J16 1701 RATITADESSSTAYLELSSLRSEDTAVYYCAR 17J16 1702 RATITADESSSTAYMELSRLRSEDTAVYYCAR 17J16 1703 RATITADESTSTAYLELSRLRSEDTAVYYCAR 17J16 1704 RVTLTADESSSTAYLELSSLRSEDTAVYYCAR 17J16 1705 RVTLTADESSSTAYMELSRLRSEDTAVYYCAR 17J16 1706 RVTLTADESTSTAYLELSRLRSEDTAVYYCAR 17J16 1707 RVTITADESSSTAYLELSRLRSEDTAVYYCAR 17J16 1708 RATLTADESSSTAYLELSSLRSEDTAVYYCAR 17J16 1709 RATLTADESSSTAYMELSRLRSEDTAVYYCAR 17J16 1710 RATLTADESTSTAYLELSRLRSEDTAVYYCAR 17J16 1711 RATITADESSSTAYLELSRLRSEDTAVYYCAR 17J16 1712 RVTLTADESSSTAYLELSRLRSEDTAVYYCAR 17J16 1713 RATLTADESSSTAYLELSRLRSEDTAVYYCAR 17J16 1714 RVTITRDTSASTAYMELSSLRSEDTAVYYCAR 5F12 1715 RATITRDESASTAYMELSSLRSEDTAVYYCAR 5F12 1716 RVTLTRDESASTAYMELSSLRSEDTAVYYCAR 5F12 1717 RVTITRDKSASTAYMELSSLRSEDTAVYYCAR 5F12 1718 RATLTRDESASTAYMELSSLRSEDTAVYYCAR 5F12 1719 RATITRDKSASTAYMELSSLRSEDTAVYYCAR 5F12 1720 RVTLTRDKSASTAYMELSSLRSEDTAVYYCAR 5F12 1721 RATLTRDKSASTAYMELSSLRSEDTAVYYCAR 5F12 1722 QVTISADKSISTAYLQWSSLKASDTAMYYCAR 25M22 1723 QATISADKSISTAYLQWSSLKASDTAMYYCAR 25M22 1724 QVTLSADKSISTAYLQWSSLKASDTAMYYCAR 25M22 1725 QATLSADKSISTAYLQWSSLKASDTAMYYCAR 25M22 1726 VH Framework 4 (FR4) WGQGTLVTVSS 1C1, 3P10, 5F12 1727 WGQGTTVTVSS 3P10, 25M22, 17J16 1728 VL Humanized Clone SEQ ID NO: VL Framework 1 (FR1) DVVMTQSPLSLPVTLGQPASISC 1C1, 3P10, 5F12, 1729 25M22, 17J16 DVVLTQSPLSLPVTLGQPASISC 1C1, 3P10, 5F12, 1730 25M22, 17J16 DVVLTQSPLSLPVTLGDPASISC 1C1 1731 DVVLTQSPLSLPVTLGDPASISC 1C1 1732 DIVMTQSPLSLPVTLGQPASISC 3P10, 5F12 1733 DIVLTQSPLSLPVTLGQPASISC 3P10, 5F12 1734 DVAMTQSPLSLPVTLGQPASISC 17J16 1735 DVALTQSPLSLPVTLGQPASISC 17J16 1736 DVVLTQTPLSLPVSPGDQASISC 1C1 1737 DIVMTQTPLSLPVSPGDQASISC 1C1 1738 DIVMTQTPLSLPVSPGDQASISC 1C1 1739 DIVMTQTPLSLPVSPGDQASISC 1C1 1740 DIELTQSPASLAVSLGQRATISC 3P10 1741 DIVLTQSPASLAVSLGQRATISC 3P10 1742 DIVMTQSPDSLAVSLGERATINC 5F12 1743 DIVLTQSPDSLAVSLGERATINC 5F12 1744 DIQMTQSPSSLSASVGDRVTITC 5F12 1745 DIQLTQSPSSLSASVGDRVTITC 5F12 1746 EIVLTQSPATLSLSPGERATLSC 5F12 1747 EIVLTQSPATLSVSPGERATLSC 5F12 1748 EIVLTQSPGTLSLSPGERATLSC 25M22 1749 EVVLTQSPGTLSLSPGERATLSC 25M22 1750 VL Framework 2 (FR2) WFQQRPGQSPRRLIY 1C1, 3P10, 5F12, 1751 25M22, 17J16 WYQQRPGQSPRRLIY 1C1, 5F12, 25M22 1752 WFQQKPGQSPRRLIY 1C1 1753 WFQQRPGQSPKRLIY 1C1, 17J16 1754 WFQQRPGQSPRLLIY 1C1, 3P10, 5F12, 1755 25M22 WYQQKPGQSPRRLIY 1C1 1756 WYQQRPGQSPKRLIY 1C1 1757 WYQQRPGQSPRLLIY 1C1, 5F12, 25M22 1758 WFQQKPGQSPKRLIY 1C1 1759 WFQQKPGQSPRLLIY 1C1 1760 WFQQRPGQSPKLLIY 1C1 1761 WYQQKPGQSPKRLIY 1C1 1762 WYQQKPGQSPRLLIY 1C1 1763 WYQQRPGQSPKLLIY 1C1 1764 WFQQKPGQSPKLLIY 1C1 1765 WYQQKPGQSPKLLIY 1C1 1766 WLQQRPGQSPRRLIY 17J16 1767 WLQQRPGQSPKRLIY 17J16 1768 WFQQRPGQSPRRLIF 25M22 1769 WYQQRPGQSPRRLIF 25M22 1770 WFQQRPGQSPRLLIF 25M22 1771 WYQQRPGQSPRLLIF 25M22 1772 WYLQKPGQSPKLLIY 1C1 1773 WYQQKPGQPPKLLIY 3P10, 5F12 1774 WYQQKPGKAPKLLIY 5F12 1775 WYQQKPGQAPRLLIY 5F12, 25M122 1776 WYQQKPGQAPRLLIF 25M122 1777 VL Framework 3 (FR3) GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 1C1, 3P10, 5F12, 1778 25M122, 17J16 GVPDRFSGSGSGADFTLKISRVEAEDVGVYYC 17J16 1779 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC 1C1, 3P10 1780 GVPDRFSGSGSRTDFTLKISRVEAEDVGVYYC 5F12 1781 GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC 5F12 1782 GVPDRFSGSGSRTDFTLTISSLQAEDVAVYYC 5F12 1783 GVPDRFSGSGSGTDFTLTISSVQAEDVAVYYC 5F12 1784 GVPDRFSGSGSRTDFTLTISSVQAEDVAVYYC 5F12 1785 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 5F12 1786 GVPSRFSGSGSGTDFTLTISSVQPEDFATYYC 5F12 1787 GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC 5F12 1788 GVPARFSGSGSGTDFTLTISSLEPEDFAVYYC 5F12 1789 GIPARFSGSGSGTDFTLTISSVEPEDFAVYYC 5F12 1790 GVPARFSGSGSGTDFTLTISSVEPEDFAVYYC 5F12 1791 GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC 25M122 1792 VL Framework 4 (FR4) FGGGTKVEIK 1C1, 3P10, 5F12, 1793 25M122, 17J16 FGSGTKLEIK 1C1, 3P10 1794

In certain embodiments, an antibody or fragment thereof described herein comprises a VH region that comprises: (1) a VH FR1 having an amino acid sequence selected from SEQ ID NOS: 570-578; (2) a VH FR2 having an amino acid sequence selected from SEQ ID NOS: 579-602; (3) a VH FR3 having an amino acid sequence selected from SEQ ID NOS: 603-1726; and/or (4) a VH FR4 having an amino acid selected from SEQ ID NOS: 1727-1728. Accordingly, in some aspects, the humanized antibody comprises a VH region that includes a VH FR1 having an amino acid sequence selected from SEQ ID NOS: 570-578. In some aspects, the humanized antibody comprises a VH region that includes a VH FR2 having an amino acid sequence selected from SEQ ID NOS: 579-602. In some aspects, the humanized antibody comprises a VH region that includes a VH FR3 having an amino acid sequence selected from SEQ ID NOS: 603-1726. In some aspects, the humanized antibody comprises a VH region that includes a VH FR4 having an amino acid selected from SEQ ID NOS: 1727-1728.

In certain embodiments, an antibody or fragment thereof described herein comprises a VL region that comprises: (1) a VL FR1 having an amino acid sequence selected from SEQ ID NOS: 1729-1750; (2) a VL FR2 having an amino acid sequence selected from SEQ ID NOS: 1751-1777; (3) a VL FR3 having an amino acid sequence selected from SEQ ID NOS: 1778-1792; and/or (4) a VL FR4 having an amino acid selected from SEQ ID NOS: 1793-1794. Accordingly, in some aspects, the humanized antibody comprises a VL region that includes a VL FR1 having an amino acid sequence selected from SEQ ID NOS: 1729-1750. In some aspects, the humanized antibody comprises a VL region that includes a VL FR2 having an amino acid sequence selected from SEQ ID NOS: 1751-1777. In some aspects, the humanized antibody comprises a VL region that includes a VL FR3 having an amino acid sequence selected from SEQ ID NOS: 1778-1792. In some aspects, the humanized antibody comprises a VL region that includes a VL FR4 having an amino acid selected from SEQ ID NOS: 1793-1794.

In certain embodiments, an antibody or fragment thereof described herein comprises a VH region and a VL region, wherein the VH region further comprises: (1) a VH FR1 having an amino acid sequence selected from SEQ ID NOS: 570-578; (2) a VH FR2 having an amino acid sequence selected from SEQ ID NOS: 579-602; (3) a VH FR3 having an amino acid sequence selected from SEQ ID NOS: 603-1726; and/or (4) a VH FR4 having an amino acid sequence of SEQ ID NOS: 1727-1728; and wherein the VL region further comprises: (1) a VL FR1 having an amino acid sequence selected from SEQ ID NOS: 1729-1750; (2) a VL FR2 having an amino acid sequence selected from SEQ ID NOS: 1751-1777; (3) a VL FR3 having an amino acid sequence selected from SEQ ID NOS: 1778-1792; and/or (4) a VL FR4 having an amino acid selected from SEQ ID NOS: 1793-1794.

Also provided herein are antibodies comprising one or more (e.g., one, two, three or four) VH FRs and one or more (e.g., one, two, three or four) VL FRs listed in Table 25. In particular, provided herein is an antibody comprising: a VH FR1 (SEQ ID NOS:570-578) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR2 (SEQ ID NOS:579-602) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR3 (SEQ ID NOS:603-1726) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR3 (SEQ ID NOS:603-1726) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR1 (SEQ ID NOS:1729-1750); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), a VL FR3 (SEQ ID NOS:1778-1792), and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750) and a VL FR2 (SEQ ID NOS:1751-1777); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), a VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR3 (SEQ ID NOS:1778-1792); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); a VH FR1 (SEQ ID NOS:570-578), a VH FR2 (SEQ ID NOS:579-602), a VH FR3 (SEQ ID NOS:603-1726), a VH FR4 (SEQ ID NOS:1727-1728), a VL FR1 (SEQ ID NOS:1729-1750), VL FR2 (SEQ ID NOS:1751-1777), VL FR3 (SEQ ID NOS:1778-1792) and a VL FR4 (SEQ ID NOS:1793-1794); or any combination thereof of the VH FRs (SEQ ID NOS: 478-1636) and VL FRs (SEQ ID NOS: 1637-1702) listed in Tables 25.

In yet another aspect, antibodies are provided that compete with one of the exemplified antibodies or functional fragments for binding to GFRAL. Such antibodies may also bind to the same epitope as one of the herein exemplified antibodies, or an overlapping epitope. Antibodies and fragments that compete with or bind to the same epitope as the exemplified antibodies are expected to show similar functional properties. The exemplified antigen binding proteins and fragments include those with the VH and VL regions, and CDRs provided herein, including those in Tables 1-24. Thus, as a specific example, the antibodies that are provided include those that compete with an antibody comprising: (a) 1, 2, 3, 4, 5 or all 6 of the CDRs listed for an antibody listed in Tables 1-24; (b) a VH and a VL selected from the VH and a VL regions listed for an antibody listed in Tables 1-24; or (c) two light chains and two heavy chains comprising a VH and a VL as specified for an antibody listed in Tables 1-24.

In certain embodiments, antibodies of the compositions and methods of using the antibodies provided herein include those anti-GFRAL monoclonal antibodies described herein, e.g., in the Examples section. Any anti-GFRAL antibody can be used in any of the methods provided herein. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 1, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 3, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 5, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 7, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 9, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 11, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 13, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 15, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 17, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 17, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 19, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 21, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 23, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 25, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 27, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 29, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 31, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 33, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 35, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 37, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 39, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 480, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 482, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 484, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 486, or a humanized variant thereof.

In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 2, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 4, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 6, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 8, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 10, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 12, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 14, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 16, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 18, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 20, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 22, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 24, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 26, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 28, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 30, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 32, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 34, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 36, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 38, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 40, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 481, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 483, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 485, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VL region comprising the amino acid sequence of SEQ ID NO: 487, or a humanized variant thereof.

In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 1, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 2, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 3, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 4, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 5, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 6, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 7, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 8, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 9, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 10, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 11, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 12, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 13, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 14, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 15, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 16, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 17, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 18, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 19, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 20, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 21, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 22, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 23, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 24, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 25, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 26, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 27, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 28, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 29, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 30, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 31, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 32, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 33, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 34, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 35, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 36, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 37, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 38, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 39, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 40, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 480, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 481, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 482, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 483, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 484, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 485, or a humanized variant thereof. In some embodiments of the various methods provided herein, the antibody comprises a VH region comprising the amino acid sequence of SEQ ID NO: 486, or a humanized variant thereof, and a VL region comprising the amino acid sequence of SEQ ID NO: 487, or a humanized variant thereof.

In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 1 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 3 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 5 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 7 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 9 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 11 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 13 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 14. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 15 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 17 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 19 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 21 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 23 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 25 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 27 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 29 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 31 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 33 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 35 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 37 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 39 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 480 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 481. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 482 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 483. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 484 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 485. In some embodiments of the various methods provided herein, the antibody comprises a VH CDR1, VH CDR2, and VH CDR3 of a VH domain having the amino acid sequence of SEQ ID NO: 486 and a VL CDR1, VL CDR2, and VL CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 487. Other VH domain, VL domain, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 sequences listed described herein are also contemplated for use in the various methods provided herein.

1. Polyclonal Antibodies

The antibodies of the present disclosure may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a GFRAL polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized or to immunize the mammal with the protein and one or more adjuvants. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Ribi, CpG, Poly 1C, Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. The mammal can then be bled, and the serum assayed for GFRAL antibody titer. If desired, the mammal can be boosted until the antibody titer increases or plateaus. Additionally or alternatively, lymphocytes may be obtained from the immunized animal for fusion and the preparation of monoclonal antibodies from hybridoma as described below.

2. Monoclonal Antibodies

The antibodies of the present disclosure may alternatively be monoclonal antibodies. Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as SP-2 and derivatives, for example, X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA and those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J., Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. The binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal, for example, by i.p. injection of the cells into mice.

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262 (1993) and Plückthun, Immunol. Revs. 130:151-188 (1992).

In some embodiments, an antibody that binds a GFRAL epitope comprises an amino acid sequence of a VH domain and/or an amino acid sequence of a VL domain encoded by a nucleotide sequence that hybridizes to (1) the complement of a nucleotide sequence encoding any one of the VH and/or VL domain described herein under stringent conditions (e.g., hybridization to filter-bound DNA in 6× sodium chloride/sodium citrate (SSC) at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.) under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).

In some embodiments, an antibody that binds a GFRAL epitope comprises an amino acid sequence of a VH CDR or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes to the complement of a nucleotide sequence encoding any one of the VH CDRs and/or VL CDRs depicted in Tables 1-24 under stringent conditions (e.g., hybridization to filter-bound DNA in 6×SSC at about 45° C. followed by one or more washes in 0.2×SSC/0.1% SDS at about 50-65° C.), under highly stringent conditions (e.g., hybridization to filter-bound nucleic acid in 6×SSC at about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 68° C.), or under other stringent hybridization conditions which are known to those of skill in the art (see, for example, Ausubel, F. M. et al., eds., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York at pages 6.3.1-6.3.6 and 2.10.3).

In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in, for example, Antibody Phage Display: Methods and Protocols, P. M. O'Brien and R. Aitken, eds, Humana Press, Totawa N.J., 2002. In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. Such phage libraries are screened for against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution.

Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described, for example, in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).

Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigen-binding clones as described in Winter et al., supra. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning the unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described, for example, by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

Screening of the libraries can be accomplished by various techniques known in the art. For example, GFRAL, (e.g., a GFRAL polypeptide, fragment or epitope) can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries. The selection of antibodies with slow dissociation kinetics (e.g., good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992).

Anti-GFRAL antibodies can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-GFRAL antibody clone using VH and/or VL sequences (e.g., the Fv sequences), or various CDR sequences from VH and VL sequences, from the phage clone of interest and suitable constant region (e.g., Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

3. Antibody Fragments

The present disclosure provides antibodies and antibody fragments that bind to GFRAL. In certain circumstances there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to cells, tissues or organs. For a review of certain antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E. coli or yeast cells, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another approach, F(ab′)2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab′)2 fragment with increased in vivo half-life comprising salvage receptor binding epitope residues are described, for example, U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In certain embodiments, an antibody is a single chain Fv fragment (scFv) (see, e.g., WO 93/16185; U.S. Pat. Nos. 5,571,894; and 5,587,458). Fv and scFv have intact combining sites that are devoid of constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv. (See, e.g., Antibody Engineering, ed. Borrebaeck, supra). The antibody fragment may also be a “linear antibody”, for example, as described, for example, in the references cited above. Such linear antibodies may be monospecific or multi-specific, such as bispecific.

Smaller antibody-derived binding structures are the separate variable domains (V domains) also termed single variable domain antibodies (SdAbs). Certain types of organisms, the camelids and cartilaginous fish, possess high affinity single V-like domains mounted on an Fc equivalent domain structure as part of their immune system. (Woolven et al., Immunogenetics 50: 98-101, 1999; Streltsov et al., Proc Natl Acad Sci USA. 101:12444-12449, 2004). The V-like domains (called VhH in camelids and V-NAR in sharks) typically display long surface loops, which allow penetration of cavities of target antigens. They also stabilize isolated VH domains by masking hydrophobic surface patches.

These VhH and V-NAR domains have been used to engineer sdAbs. Human V domain variants have been designed using selection from phage libraries and other approaches that have resulted in stable, high binding VL- and VH-derived domains.

Antibodies that bind to GFRAL as provided herein include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments, (e.g., GFRAL binding fragments) of any of the above. Non-limiting examples of functional fragments (e.g., fragments that bind to GFRAL) include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab′) fragments, F(ab)2 fragments, F(ab′)2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody.

Antibodies provided herein include, but are not limited to, immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, for example, molecules that contain an antigen binding site that bind to a GFRAL epitope. The immunoglobulin molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Variants and derivatives of antibodies include antibody functional fragments that retain the ability to bind to a GFRAL epitope. Exemplary functional fragments include Fab fragments (e.g., an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab′ (e.g., an antibody fragment containing a single anti-binding domain comprising an Fab and an additional portion of the heavy chain through the hinge region); F(ab′)2 (e.g., two Fab′ molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab′ molecules may be directed toward the same or different epitopes); a bispecific Fab (e.g., a Fab molecule having two antigen binding domains, each of which may be directed to a different epitope); a single chain Fab chain comprising a variable region, also known as, a sFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); a disulfide-linked Fv, or dsFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a camelized VH (e.g., the variable, antigen-binding determinative region of a single heavy chain of an antibody in which some amino acids at the VH interface are those found in the heavy chain of naturally occurring camel antibodies); a bispecific sFv (e.g., a sFv or a dsFv molecule having two antigen-binding domains, each of which may be directed to a different epitope); a diabody (e.g., a dimerized sFv formed when the VH domain of a first sFv assembles with the VL domain of a second sFv and the VL domain of the first sFv assembles with the VH domain of the second sFv; the two antigen-binding regions of the diabody may be directed towards the same or different epitopes); and a triabody (e.g., a trimerized sFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen binding domains may be directed towards the same or different epitopes). Derivatives of antibodies also include one or more CDR sequences of an antibody combining site. The CDR sequences may be linked together on a scaffold when two or more CDR sequences are present. In certain embodiments, the antibody comprises a single-chain Fv (“scFv”). scFvs are antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The scFv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

4. Humanized Antibodies

The present disclosure provides humanized antibodies that bind GFRAL, including human GFRAL. Humanized antibodies of the present disclosure may comprise one or more CDRs as shown in Tables 1-24. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody can have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization may be performed, for example, following the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody.

In some cases, the humanized antibodies are constructed by CDR grafting, in which the amino acid sequences of the six complementarity determining regions (CDRs) of the parent non-human antibody (e.g., rodent) are grafted onto a human antibody framework. For example, Padlan et al. (FASEB J. 9:133-139, 1995) determined that only about one third of the residues in the CDRs actually contact the antigen, and termed these the “specificity determining residues,” or SDRs. In the technique of SDR grafting, only the SDR residues are grafted onto the human antibody framework (see, e.g., Kashmiri et al., Methods 36: 25-34, 2005).

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies can be important to reduce antigenicity. For example, according to the so-called “best-fit” method, the sequence of the variable domain of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent may be selected as the human framework for the humanized antibody (Sims et al. (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623. In some cases, the framework is derived from the consensus sequences of the most abundant human subclasses, V_(L)6 subgroup I (V_(L)6I) and VH subgroup III (V_(H)III). In another method, human germline genes are used at the source of the framework regions.

In an alternative paradigm based on comparison of CDRs, called Superhumanization, FR homology is irrelevant. The method consists of comparison of the non-human sequence with the functional human germline gene repertoire. Those genes encoding the same or closely related canonical structures to the murine sequences are then selected. Next, within the genes sharing the canonical structures with the non-human antibody, those with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these FRs (see, e.g., Tan et al., J. Immunol. 169: 1119-1125, 2002).

It is further generally desirable that antibodies be humanized with retention of their affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. These include, for example, WAM (Whitelegg and Rees, Protein Eng. 13: 819-824, 2000), Modeller (Sali and Blundell, J. Mol. Biol. 234: 779-815, 1993), and Swiss PDB Viewer (Guex and Peitsch, Electrophoresis 18: 2714-2713, 1997). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

Another method for antibody humanization is based on a metric of antibody humanness termed Human String Content (HSC). This method compares the mouse sequence with the repertoire of human germline genes and the differences are scored as HSC. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants. (Lazar et al., Mol. Immunol. 44: 1986-1998, 2007).

In addition to the methods described above, empirical methods may be used to generate and select humanized antibodies. These methods include those that are based upon the generation of large libraries of humanized variants and selection of the best clones using enrichment technologies or high throughput screening techniques. Antibody variants may be isolated from phage, ribosome and yeast display libraries as well as by bacterial colony screening (see, e.g., Hoogenboom, Nat. Biotechnol. 23: 1105-1116, 2005; Dufner et al., Trends Biotechnol. 24: 523-529, 2006; Feldhaus et al., Nat. Biotechnol. 21: 163-70, 2003; Schlapschy et al., Protein Eng. Des. Sel. 17: 847-60, 2004).

In the FR library approach, a collection of residue variants are introduced at specific positions in the FR followed by selection of the library to select the FR that best supports the grafted CDR. The residues to be substituted may include some or all of the “Vernier” residues identified as potentially contributing to CDR structure (see, e.g., Foote and Winter, J. Mol. Biol. 224: 487-499, 1992), or from the more limited set of target residues identified by Baca et al. (J. Biol. Chem. 272: 10678-10684, 1997).

In FR shuffling, whole FRs are combined with the non-human CDRs instead of creating combinatorial libraries of selected residue variants (see, e.g., Dall'Acqua et al., Methods 36: 43-60, 2005). The libraries may be screened for binding in a two-step selection process, first humanizing VL, followed by VH. Alternatively, a one-step FR shuffling process may be used. Such a process has been shown to be more efficient than the two-step screening, as the resulting antibodies exhibited improved biochemical and physico-chemical properties including enhanced expression, increased affinity and thermal stability (see, e.g., Damschroder et al., Mol. Immunol. 44: 3049-60, 2007).

The “humaneering” method is based on experimental identification of essential minimum specificity determinants (MSDs) and is based on sequential replacement of non-human fragments into libraries of human FRs and assessment of binding. It begins with regions of the CDR3 of non-human VH and VL chains and progressively replaces other regions of the non-human antibody into the human FRs, including the CDR1 and CDR2 of both VH and VL. This methodology typically results in epitope retention and identification of antibodies from multiple sub-classes with distinct human V-segment CDRs. Humaneering allows for isolation of antibodies that are 91-96% homologous to human germline gene antibodies. (see, e.g., Alfenito, Cambridge Healthtech Institute's Third Annual PEGS, The Protein Engineering Summit, 2007).

The “human engineering” method involves altering an non-human antibody or antibody fragment, such as a mouse or chimeric antibody or antibody fragment, by making specific changes to the amino acid sequence of the antibody so as to produce a modified antibody with reduced immunogenicity in a human that nonetheless retains the desirable binding properties of the original non-human antibodies. Generally, the technique involves classifying amino acid residues of a non-human (e.g., mouse) antibody as “low risk”, “moderate risk”, or “high risk” residues. The classification is performed using a global risk/reward calculation that evaluates the predicted benefits of making particular substitution (e.g., for immunogenicity in humans) against the risk that the substitution will affect the resulting antibody's folding and/or are substituted with human residues. The particular human amino acid residue to be substituted at a given position (e.g., low or moderate risk) of a non-human (e.g., mouse) antibody sequence can be selected by aligning an amino acid sequence from the non-human antibody's variable regions with the corresponding region of a specific or consensus human antibody sequence. The amino acid residues at low or moderate risk positions in the non-human sequence can be substituted for the corresponding residues in the human antibody sequence according to the alignment. Techniques for making human engineered proteins are described in greater detail in Studnicka et al., Protein Engineering, 7: 805-814 (1994), U.S. Pat. Nos. 5,766,886, 5,770,196, 5,821,123, and 5,869,619, and PCT Application Publication WO 93/11794.

5. Human Antibodies

Human anti-GFRAL antibodies can be constructed by combining Fv clone variable domain sequence(s) selected from human-derived phage display libraries with known human constant domain sequences(s). Alternatively, human monoclonal GFRAL antibodies of the present disclosure can be made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is also possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. Transgenic mice that express human antibody repertoires have been used to generate high-affinity human sequence monoclonal antibodies against a wide variety of potential drug targets (see, e.g., Jakobovits, A., Curr. Opin. Biotechnol. 1995, 6(5):561-6; Brüggemann and Taussing, Curr. Opin. Biotechnol. 1997, 8(4):455-8; U.S. Pat. Nos. 6,075,181 and 6,150,584; and Lonberg et al., Nature Biotechnol. 23: 1117-1125, 2005).

Alternatively, the human antibody may be prepared via immortalization of human B lymphocytes producing an antibody directed against a target antigen (e.g., such B lymphocytes may be recovered from an individual or may have been immunized in vitro) (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373).

Gene shuffling can also be used to derive human antibodies from non-human, for example, rodent, antibodies, where the human antibody has similar affinities and specificities to the starting non-human antibody. According to this method, which is also called “epitope imprinting” or “guided selection”, either the heavy or light chain variable region of a non-human antibody fragment obtained by phage display techniques as described herein is replaced with a repertoire of human V domain genes, creating a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in isolation of a non-human chain/human chain chimeric scFv or Fab wherein the human chain restores the antigen binding site destroyed upon removal of the corresponding non-human chain in the primary phage display clone, (e.g., the epitope guides (imprints) the choice of the human chain partner). When the process is repeated in order to replace the remaining non-human chain, a human antibody is obtained (see, e.g., PCT WO 93/06213; and Osbourn et al., Methods., 36, 61-68, 2005). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides completely human antibodies, which have no FR or CDR residues of non-human origin. Examples of guided selection to humanize mouse antibodies towards cell surface antigens include the folate-binding protein present on ovarian cancer cells (see, e.g., Figini et al., Cancer Res., 58, 991-996, 1998) and CD147, which is highly expressed on hepatocellular carcinoma (see, e.g., Bao et al., Cancer Biol. Ther., 4, 1374-1380, 2005).

A potential disadvantage of the guided selection approach is that shuffling of one antibody chain while keeping the other constant could result in epitope drift. In order to maintain the epitope recognized by the non-human antibody, CDR retention can be applied (see, e.g., Klimka et al., Br. J. Cancer., 83, 252-260, 2000; VH CDR2 Beiboer et al., J. Mol. Biol., 296, 833-49, 2000) In this method, the non-human VH CDR3 is commonly retained, as this CDR may be at the center of the antigen-binding site and may be to be the most important region of the antibody for antigen recognition. In some instances, however, VH CDR3 and VL CDR3, as well as VH CDR3, VL CDR3 and VL CFR1, of the non-human antibody may be retained.

6. Bispecific Antibodies

Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different antigens. In certain embodiments, bispecific antibodies are human or humanized antibodies. In certain embodiments, one of the binding specificities is for GFRAL and the other is for any other antigen. In some embodiments, bispecific antibodies can bind to two different epitopes of GFRAL. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art, such as, for example, by co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (see, e.g., Milstein and Cuello, Nature, 305: 537 (1983)). For further details of generating bispecific antibodies see, for example, Bispecific Antibodies, Kontermann, ed., Springer-Verlag, Hiedelberg (2011).

7. Multivalent Antibodies

A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present disclosure can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. In certain embodiments, the dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. In certain embodiments, a multivalent antibody comprises (or consists of) three to about eight antigen binding sites. In one such embodiment, a multivalent antibody comprises (or consists of) four antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (for example, two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains. For instance, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein may further comprise at least two (for example, four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.

8. Fc Engineering

It may be desirable to modify an antibody to GFRAL via Fc engineering, including, with respect to effector function, for example, so as to decrease or remove antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. For example, substitutions into human IgG1 using IgG2 residues as positions 233-236 and IgG4 residues at positions 327, 330 and 331 were shown to greatly reduce ADCC and CDC (see, e.g., Armour et al., Eur. J. Immunol. 29:(8):2613-24 (1999); Shields et al., J. Biol. Chem. 276(9): 6591-604 (2001).

To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment), for example, as described in U.S. Pat. No. 5,739,277. Term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

9. Alternative Binding Agents

The present disclosure encompasses non-immunoglobulin binding agents that specifically bind to the same epitope as an anti-GFRAL antibody disclosed herein. In some embodiments, a non-immunoglobulin binding agent is identified an agent that displaces or is displaced by an anti-GFRAL antibody of the present disclosure in a competive binding assay. These alternative binding agents may include, for example, any of the engineered protein scaffolds known in the art. Such scaffolds may comprise one or more CDRs as shown in Tables 1-24. Such scaffolds include, for example, anticalins, which are based upon the lipocalin scaffold, a protein structure characterized by a rigid beta-barrel that supports four hypervariable loops which form the ligand binding site. Novel binding specificities may be engineered by targeted random mutagenesis in the loop regions, in combination with functional display and guided selection (see, e.g., Skerra (2008) FEBS J. 275: 2677-2683). Other suitable scaffolds may include, for example, adnectins, or monobodies, based on the tenth extracellular domain of human fibronectin III (see, e.g., Koide and Koide (2007) Methods Mol. Biol. 352: 95-109); affibodies, based on the Z domain of staphylococcal protein A (see, e.g., Nygren et al. (2008) FEBS J. 275: 2668-2676)); DARPins, based on ankyrin repeat proteins (see, e.g., Stumpp et al. (2008) Drug. Discov. Today 13: 695-701); fynomers, based on the SH3 domain of the human Fyn protein kinase Grabulovski et al. (2007) J. Biol. Chem. 282: 3196-3204); affitins, based on Sac7d from Sulfolobus acidolarius (see, e.g., Krehenbrink et al. (2008) J. Mol. Biol. 383: 1058-1068); affilins, based on human y-B-crystallin (see, e.g., Ebersbach et al. (2007) J. Mol. Biol. 372: 172-185); avimers, based on the A domains of membrane receptor proteins (see, e.g., Silverman et al. (2005) Biotechnol. 23: 1556-1561); cysteine-rich knottin peptides (see, e.g., Kolmar (2008) FEBS J. 275: 2684-2690); and engineered Kunitz-type inhibitors (see, e.g., Nixon and Wood (2006) Curr. Opin. Drug. Discov. Dev. 9: 261-268) For a review, see, for example, Gebauer and Skerra (2009) Curr. Opin. Chem. Biol. 13: 245-255.

Antibody Variants

In some embodiments, amino acid sequence modification(s) of the antibodies that bind to GFRAL described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody, including but not limited to specificity, thermostability, expression level, effector functions, glycosylation, reduced immunogenicity or solubility. This, in addition to the anti-GFRAL antibodies described herein, it is contemplated that anti-GFRAL antibody variants can be prepared. For example, anti-GFRAL antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the anti-GFRAL antibody, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

In some embodiments, antibodies provided herein are chemically modified, for example, by the association with, including covalent attachment of, any type of molecule with the antibody. The antibody derivatives may include antibodies that have been chemically modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited, to specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. Additionally, the antibody may contain one or more non-classical amino acids.

Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In certain embodiments, the substitution, deletion or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule. In a specific embodiment, the substitution is a conservative amino acid substitution made at one or more predicted non-essential amino acid residues. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Alternatively, conservative (e.g., within an amino acid group with similar properties and/or sidechains) substitutions may be made, so as to maintain or not significantly change the properties. Amino acids may be grouped according to similarities in the properties of their side chains (see, e.g., A. L. Lehninger, in Biochemistry, 2nd Ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); and (4) basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, into the remaining (non-conserved) sites. Accordingly, in one embodiment, an antibody or fragment thereof that binds to a GFRAL epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a murine monoclonal antibody described herein. In one embodiment, an antibody or fragment thereof that binds to a GFRAL epitope comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to an amino acid sequence depicted in Tables 1-24. In yet another embodiment, an antibody or fragment thereof that binds to a GFRAL epitope comprises a VH CDR and/or a VL CDR amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a VH CDR amino acid sequence depicted in Tables 1-24 and/or a VL CDR amino acid sequence depicted in Tables 1-24. The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (see, e.g., Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the anti-GFRAL antibody variant DNA.

Any cysteine residue not involved in maintaining the proper conformation of the anti-GFRAL antibody also may be substituted, for example, with another amino acid such as alanine or serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the anti-GFRAL antibody to improve its stability (e.g., where the antibody is an antibody fragment such as an Fv fragment).

In some embodiments, an anti-GFRAL antibody molecule of the present disclosure is a “de-immunized” antibody. A “de-immunized” anti-GFRAL antibody is an antibody derived from a humanized or chimeric anti-GFRAL antibody, that has one or more alterations in its amino acid sequence resulting in a reduction of immunogenicity of the antibody, compared to the respective original non-de-immunized antibody. One of the procedures for generating such antibody mutants involves the identification and removal of T-cell epitopes of the antibody molecule. In a first step, the immunogenicity of the antibody molecule can be determined by several methods, for example, by in vitro determination of T-cell epitopes or in silico prediction of such epitopes, as known in the art. Once the critical residues for T-cell epitope function have been identified, mutations can be made to remove immunogenicity and retain antibody activity. For review, see, for example, Jones et al., Methods in Molecular Biology 525: 405-423, 2009.

1. In Vitro Affinity Maturation

In some embodiments, antibody variants having an improved property such as affinity, stability, or expression level as compared to a parent antibody may be prepared by in vitro affinity maturation. Like the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. Libraries of antibodies are displayed as Fab, scFv or V domain fragments either on the surface of an organism (e.g., phage, bacteria, yeast or mammalian cell) or in association (e.g., covalently or non-covalently) with their encoding mRNA or DNA. Affinity selection of the displayed antibodies allows isolation of organisms or complexes carrying the genetic information encoding the antibodies. Two or three rounds of mutation and selection using display methods such as phage display usually results in antibody fragments with affinities in the low nanomolar range. Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen.

Phage display is a widespread method for display and selection of antibodies. The antibodies are displayed on the surface of Fd or M13 bacteriophages as fusions to the bacteriophage coat protein. Selection involves exposure to antigen to allow phage-displayed antibodies to bind their targets, a process referred to as “panning.” Phage bound to antigen are recovered and infected in bacteria to produce phage for further rounds of selection. For review, see, for example, Hoogenboom, Methods. Mol. Biol. 178: 1-37, 2002; Bradbury and Marks, J. Immuno. Methods 290: 29-49, 2004).

In a yeast display system (see, e.g., Boder et al., Nat. Biotech. 15: 553-57, 1997; Chao et al., Nat. Protocols 1:755-768, 2006), the antibody may be displayed as single-chain variable fusions (scFv) in which the heavy and light chains are connected by a flexible linker. The scFv is fused to the adhesion subunit of the yeast agglutinin protein Aga2p, which attaches to the yeast cell wall through disulfide bonds to Aga1p. Display of a protein via Aga2p projects the protein away from the cell surface, minimizing potential interactions with other molecules on the yeast cell wall. Magnetic separation and flow cytometry are used to screen the library to select for antibodies with improved affinity or stability. Binding to a soluble antigen of interest is determined by labeling of yeast with biotinylated antigen and a secondary reagent such as streptavidin conjugated to a fluorophore. Variations in surface expression of the antibody can be measured through immunofluorescence labeling of either the hemagglutinin or c-Myc epitope tag flanking the scFv. Expression has been shown to correlate with the stability of the displayed protein, and thus antibodies can be selected for improved stability as well as affinity (see, e.g., Shusta et al., J. Mol. Biol. 292: 949-956, 1999). An additional advantage of yeast display is that displayed proteins are folded in the endoplasmic reticulum of the eukaryotic yeast cells, taking advantage of endoplasmic reticulum chaperones and quality-control machinery. Once maturation is complete, antibody affinity can be conveniently ‘titrated’ while displayed on the surface of the yeast, eliminating the need for expression and purification of each clone. A theoretical limitation of yeast surface display is the potentially smaller functional library size than that of other display methods; however, a recent approach uses the yeast cells' mating system to create combinatorial diversity estimated to be 10¹⁴ in size (see, e.g., US Patent Publication 2003/0186,374; Blaise et al., Gene 342: 211-218, 2004).

In ribosome display, antibody-ribosome-mRNA (ARM) complexes are generated for selection in a cell-free system. The DNA library coding for a particular library of antibodies is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The resulting complex of mRNA, ribosome, and protein can bind to surface-bound ligand, allowing simultaneous isolation of the antibody and its encoding mRNA through affinity capture with the ligand. The ribosome-bound mRNA is then reversed transcribed back into cDNA, which can then undergo mutagenesis and be used in the next round of selection (see, e.g., Fukuda et al., Nucleic Acids Res. 34, e127, 2006). In mRNA display, a covalent bond between antibody and mRNA is established using puromycin as an adaptor molecule (Wilson et al., Proc. Natl. Acad. Sci. USA 98, 3750-3755, 2001).

As these methods are performed entirely in vitro, they provide two main advantages over other selection technologies. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, for example, by non-proofreading polymerases, as no library must be transformed after any diversification step.

Diversity may be introduced into the CDRs or the whole V genes of the antibody libraries in a targeted manner or via random introduction. The former approach includes sequentially targeting all the CDRs of an antibody via a high or low level of mutagenesis or targeting isolated hot spots of somatic hypermutations (see, e.g., Ho, et al., J. Biol. Chem. 280: 607-617, 2005) or residues suspected of affecting affinity on experimental basis or structural reasons. Random mutations can be introduced throughout the whole V gene using E. coli mutator strains, error-prone replication with DNA polymerases (see, e.g., Hawkins et al., J. Mol. Biol. 226: 889-896, 1992) or RNA replicases. Diversity may also be introduced by replacement of regions that are naturally diverse via DNA shuffling or similar techniques (see, e.g., Lu et al., J. Biol. Chem. 278: 43496-43507, 2003; U.S. Pat. No. 5,565,332; 6,989,250). Alternative techniques target hypervariable loops extending into framework-region residues (see, e.g., Bond et al., J. Mol. Biol. 348: 699-709, 2005) employ loop deletions and insertions in CDRs or use hybridization-based diversification (see, e.g., US Patent Publication No. 2004/0005709). Additional methods of generating diversity in CDRs are disclosed, for example, in U.S. Pat. No. 7,985,840.

Screening of the libraries can be accomplished by various techniques known in the art. For example, GFRAL can be immobilized onto solid supports, columns, pins or cellulose/poly(vinylidene fluoride) membranes/other filters, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning display libraries.

For review of in vitro affinity maturation methods, see, e.g., Hoogenboom, Nature Biotechnology 23: 1105-1116, 2005 and Quiroz and Sinclair, Revista Ingeneria Biomedia 4: 39-51, 2010 and references therein.

2. Modifications of Anti-GFRAL Antibodies

Covalent modifications of anti-GFRAL antibodies are included within the scope of the present disclosure. Covalent modifications include reacting targeted amino acid residues of an anti-GFRAL antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the anti-GFRAL antibody. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (see, e.g., T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Other types of covalent modification of the anti-GFRAL antibody included within the scope of this present disclosure include altering the native glycosylation pattern of the antibody or polypeptide (see, e.g., Beck et al., Curr. Pharm. Biotechnol. 9: 482-501, 2008; Walsh, Drug Discov. Today 15: 773-780, 2010), and linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth, for example, in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

An anti-GFRAL antibody of the present disclosure may also be modified to form chimeric molecules comprising an anti-GFRAL antibody fused to another, heterologous polypeptide or amino acid sequence, for example, an epitope tag (see, e.g., Terpe, Appl. Microbiol. Biotechnol. 60: 523-533, 2003) or the Fc region of an IgG molecule (see, e.g., Aruffo, “Immunoglobulin fusion proteins” in Antibody Fusion Proteins, S. M. Chamow and A. Ashkenazi, eds., Wiley-Liss, New York, 1999, pp. 221-242).

Also provided herein are fusion proteins comprising an antibody provided herein that binds to a GFRAL antigen and a heterologous polypeptide. In some embodiments, the heterologous polypeptide to which the antibody is fused is useful for targeting the antibody to cells having cell surface-expressed GFRAL.

Also provided herein are panels of antibodies that bind to a GFRAL antigen. In specific embodiments, panels of antibodies have different association rate constants different dissociation rate constants, different affinities for GFRAL antigen, and/or different specificities for a GFRAL antigen. In some embodiments, the panels comprise or consist of about 10, about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, or about 1000 antibodies or more. Panels of antibodies can be used, for example, in 96 well or 384 well plates, such as for assays such as ELISAs.

Preparation of Anti-GFRAL Antibodies

Anti-GFRAL antibodies may be produced by culturing cells transformed or transfected with a vector containing anti-GFRAL antibody-encoding nucleic acids. Polynucleotide sequences encoding polypeptide components of the antibody of the present disclosure can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridomas cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in host cells. Many vectors that are available and known in the art can be used for the purpose of the present disclosure. Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Host cells suitable for expressing antibodies of the present disclosure include prokaryotes such as Archaebacteria and Eubacteria, including Gram-negative or Gram-positive organisms, eukaryotic microbes such as filamentous fungi or yeast, invertebrate cells such as insect or plant cells, and vertebrate cells such as mammalian host cell lines. Host cells are transformed with the above-described expression vectors and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Antibodies produced by the host cells are purified using standard protein purification methods as known in the art.

Methods for antibody production including vector construction, expression and purification are further described, in Plückthun et al., (1996) in Antibody Engineering: Producing antibodies in Escherichia coli: From PCR to fermentation (McCafferty, J., Hoogenboom, H. R., and Chiswell, D. J., eds), 1 Ed., pp. 203-252, IRL Press, Oxford; Kwong, K. & Rader, C., E. coli expression and purification of Fab antibody fragments, Current protocols in protein science editorial board John E Coligan et al., Chapter 6, Unit 6.10 (2009); Tachibana and Takekoshi, “Production of Antibody Fab Fragments in Escherischia coli,” in Antibody Expression and Production, M. Al-Rubeai, Ed., Springer, New York, 2011; Therapeutic Monoclonal Antibodies: From Bench to Clinic (ed Z. An), John Wiley & Sons, Inc., Hoboken, N.J., USA.

It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-GFRAL antibodies. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)). In vitro protein synthesis may be performed using manual techniques or by automation. Various portions of the anti-GFRAL antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-GFRAL antibody. Alternatively, antibodies may be purified from cells or bodily fluids, such as milk, of a transgenic animal engineered to express the antibody, as disclosed, for example, in U.S. Pat. Nos. 5,545,807 and 5,827,690.

Immunoconjugates

The present disclosure also provides conjugates comprising any one of the anti-GFRAL antibodies of the present disclosure covalently bound, including by a synthetic linker, to one or more non-antibody agents.

A variety of radioactive isotopes are available for the production of radioconjugated antibodies. Examples include At²¹¹, I4, I4, Y4, Re4, Re4, Sm4, Bi4, P4, Pb4 and radioactive isotopes of Lu. When the conjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc4 or 14, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. The radioisotopes may be incorporated in the conjugate in known ways as described, e.g., in Reilly, “The radiochemistry of monoclonal antibodies and peptides,” in Monoclonal Antibody and Peptide-Targeted Radiotherapy of Cancer, R. M. Reilly, ed., Wiley, Hoboken N.J., 2010.

In some embodiments, antibodies provided herein are conjugated or recombinantly fused to a diagnostic, detectable or therapeutic agent or any other molecule. The conjugated or recombinantly fused antibodies can be useful, for example, for monitoring or prognosing the onset, development, progression and/or severity of a β-cell defective disease, disorder or condition as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.

Such diagnosis and detection can accomplished, for example, by coupling the antibody to detectable substances including, but not limited to, various enzymes, such as, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials, such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as, but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; chemiluminescent material, such as but not limited to, an acridinium based compound or a HALOTAG; radioactive materials, such as, but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, and ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In), technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; and positron emitting metals using various positron emission tomographies, and non-radioactive paramagnetic metal ions.

Also provided herein are antibodies that are conjugated or recombinantly fused to a therapeutic moiety (or one or more therapeutic moieties), as well as uses thereof. The antibody may be conjugated or recombinantly fused to a therapeutic moiety, including a cytotoxin such as a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion such as alpha-emitters. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.

Further, an antibody provided herein may be conjugated or recombinantly fused to a therapeutic moiety or drug moiety that modifies a given biological response. Therapeutic moieties or drug moieties are not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein, peptide, or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, γ-interferon, α-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-γ, TNF-γ, AIM I (see, e.g., International Publication No. WO 97/33899), AIM II (see, e.g., International Publication No. WO 97/34911), Fas Ligand (see, e.g., Takahashi et al., 1994, J. Immunol., 6:1567-1574), and VEGF (see, e.g., International Publication No. WO 99/23105), an anti-angiogenic agent, including, for example angiostatin, endostatin or a component of the coagulation pathway (e.g., tissue factor); or, a biological response modifier such as, for example, a lymphokine (e.g., interferon gamma, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-5 (“IL-5”), interleukin-6 (“IL-6”), interleukin-7 (“IL-7”), interleukin 9 (“IL-9”), interleukin-10 (“IL-10”), interleukin-12 (“IL-12”), interleukin-15 (“IL-15”), interleukin-23 (“IL-23”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or a growth factor (e.g., growth hormone (“GH”)), or a coagulation agent (e.g., calcium, vitamin K, tissue factors, such as but not limited to, Hageman factor (factor XII), high-molecular-weight kininogen (HMWK), prekallikrein (PK), coagulation proteins-factors II (prothrombin), factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, and fibrin monomer).

Also provided herein are antibodies that are recombinantly fused or chemically conjugated (covalent or non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, for example, to a polypeptide of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 amino acids) to generate fusion proteins, as well as uses thereof. In particular, provided herein are fusion proteins comprising an antigen-binding fragment of an antibody provided herein (e.g., a Fab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment, a VH domain, a VH CDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide, or peptide. In one embodiment, the heterologous protein, polypeptide, or peptide that the antibody is fused to is useful for targeting the antibody to a particular cell type, such as a cell that expresses GFRAL. For example, an antibody that binds to a cell surface receptor expressed by a particular cell type (e.g., an immune cell) may be fused or conjugated to a modified antibody provided herein.

In addition, an antibody provided herein can be conjugated to therapeutic moieties such as a radioactive metal ion, such as alpha-emitters such as ²¹³Bi or macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ¹³¹In, ¹³¹LU, ¹³¹Y, ¹³¹Ho, ¹³¹Sm, to polypeptides. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule. Such linker molecules are commonly known in the art and described, for example, in Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50.

Moreover, antibodies provided herein can be fused to marker or “tag” sequences, such as a peptide to facilitate purification. In specific embodiments, the marker or tag amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (see, e.g., QIAGEN, Inc.), among others, many of which are commercially available. For example, as described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the “FLAG” tag.

Methods for fusing or conjugating therapeutic moieties (including polypeptides) to antibodies are well known, (see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol. Rev. 62:119-58; U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813; Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991; Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol., 154:5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA, 89:11337-11341, 1992).

Fusion proteins may be generated, for example, through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to alter the activities of anti-GFRAL antibodies as provided herein, including, for example, antibodies with higher affinities and lower dissociation rates (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313). Antibodies, or the encoded antibodies, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. A polynucleotide encoding an antibody provided herein may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

An antibody provided herein can also be conjugated to a second antibody to form an antibody heteroconjugate as described, for example, in U.S. Pat. No. 4,676,980.

The therapeutic moiety or drug conjugated or recombinantly fused to an antibody provided herein that binds to GFRAL (e.g., a GFRAL polypeptide, fragment, epitope) should be chosen to achieve the desired prophylactic or therapeutic effect(s). In certain embodiments, the antibody is a modified antibody. A clinician or other medical personnel may consider, for example, the following when deciding on which therapeutic moiety or drug to conjugate or recombinantly fuse to an antibody provided herein: the nature of the disease, the severity of the disease, and the condition of the subject.

Antibodies that bind to GFRAL as provided herein may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

The linker may be a “cleavable linker” facilitating release of the conjugated agent in the cell, but non-cleavable linkers are also contemplated herein. Linkers for use in the conjugates of the present disclosure include without limitation acid labile linkers (e.g., hydrazone linkers), disulfide-containing linkers, peptidase-sensitive linkers (e.g., peptide linkers comprising amino acids, for example, valine and/or citrulline such as citrulline-valine or phenylalanine-lysine), photolabile linkers, dimethyl linkers (see, e.g., Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020), thioether linkers, or hydrophilic linkers designed to evade multidrug transporter-mediated resistance (see, e.g., Kovtun et al., Cancer Res. 70: 2528-2537, 2010).

Conjugates of the antibody and agent may be made using a variety of bifunctional protein coupling agents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate). The present disclosure further contemplates that conjugates of antibodies and agents may be prepared using any suitable methods as disclosed in the art, (see, e.g., in Bioconjugate Techniques, 2nd Ed., G. T. Hermanson, ed., Elsevier, San Francisco, 2008).

Conventional conjugation strategies for antibodies and agents have been based on random conjugation chemistries involving the ε-amino group of Lys residues or the thiol group of Cys residues, which results in heterogenous conjugates. Recently developed techniques allow site-specific conjugation to antibodies, resulting in homogeneous loading and avoiding conjugate subpopulations with altered antigen-binding or pharmacokinetics. These include engineering of “thiomabs” comprising cysteine substitutions at positions on the heavy and light chains that provide reactive thiol groups and do not disrupt immunoglobulin folding and assembly or alter antigen binding (see, e.g., Junutula et al., J. Immunol. Meth. 332: 41-52 (2008); Junutula et al., Nat. Biotechnol. 26: 925-932, 2008). In another method, selenocysteine is cotranslationally inserted into an antibody sequence by recoding the stop codon UGA from termination to selenocysteine insertion, allowing site specific covalent conjugation at the nucleophilic selenol group of selenocysteine in the presence of the other natural amino acids (see, e.g., Hofer et al., Proc. Natl. Acad. Sci. USA 105: 12451-12456 (2008); Hofer et al., Biochemistry 48(50): 12047-12057, 2009).

Pharmaceutical Formulations

Anti-GFRAL antibodies of the present disclosure may be administered by any route appropriate to the disease, disorder or condition to be treated. The antibody will typically be administered parenterally, for example, infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural. The antibody dose will vary, including depending on the nature and/or severity of the disease or disorder as well as the condition of the subject, may include doses between 1 mg and 100 mg. Doses may also include those between 1 mg/kg and 15 mg/kg. In some embodiments, the dose is between about 5 mg/kg and about 7.5 mg/kg. In some embodiments, the dose is about 5 mg/kg. In some embodiments, the dose is about 7.5 mg/kg. Flat doses selected from the group consisting of: (a) 375-400 mg every two weeks and (b) 550-600 mg every three weeks. In some embodiments, the flat dose is 375-400 mg every two weeks. In some embodiments, the flat dose is 550-600 mg every three weeks. In some embodiments the flat dose is 400 mg every two weeks. In some embodiments the flat dose is 600 mg every three weeks. In some embodiments of sequential dosing, a first dose and a second dose are each between 1 mg/kg and 15 mg/kg with the second dose following the first does by between 1 and 4 weeks. In some embodiments, the first dose and the second dose are each between 5 mg/kg and 7.5 mg/kg and the second dose follows the first dose by between 2 and 3 weeks. In some embodiments, the first dose and the second dose are each 5 mg/kg and the second dose follows the first dose by 2 weeks. In some embodiments, the first dose and the second dose are each 7.5 mg/kg and the second dose follows the first dose by 3 weeks.

For treating diseases, disorders or conditions, the antibody in some embodiments is administered via intravenous infusion. The dosage administered via infusion is in the range of about 1 μg/m² to about 10,000 μg/m² per dose, generally one dose per week for a total of one, two, three or four doses. Alternatively, the dosage range is of about 1 μg/m² to about 1000 μg/m², about 1 μg/m² to about 800 μg/m², about 1 μg/m² to about 600 μg/m², about 1 μg/m² to about 400 μg/m²; alternatively, about 10 μg/m² to about 500 μg/m², about 10 μg/m² to about 300 μg/m², about 10 μg/m² to about 200 μg/m², and about 1 μg/m² to about 200 μg/m². The dose may be administered once per day, once per week, multiple times per week, but less than once per day, multiple times per month but less than once per day, multiple times per month but less than once per week, once per month or intermittently to relieve or alleviate symptoms of the disease, disorder, or condition. Administration may continue at any of the disclosed intervals until amelioration of the disease, disorder or condition, or amelioration of symptoms of the disease, disorder or condition being treated. Administration may continue after remission or relief of symptoms is achieved where such remission or relief is prolonged by such continued administration.

In one aspect, the present disclosure further provides pharmaceutical formulations comprising at least one anti-GFRAL antibody of the present disclosure. In some embodiments, a pharmaceutical formulation comprises 1) an anti-GFRAL antibody, and 2) a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical formulation comprises 1) an anti-GFRAL antibody and/or an immunoconjugate thereof, and optionally, 2) at least one additional therapeutic agent.

Pharmaceutical formulations comprising an antibody is prepared for storage by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (see, e.g., Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of aqueous solutions or lyophilized or other dried formulations. The formulations herein may also contain more than one active compound as necessary for the particular disease, disorder or condition (e.g., a particular indication) being treated, preferably those with complementary activities that do not adversely affect each other. For example, in addition to an anti-GFRAL antibody, it may be desirable to include in the one formulation, an additional antibody, e.g., a second anti-GFRAL antibody which binds a different epitope on the GFRAL polypeptide, or an antibody to some other target. Alternatively, or additionally, the composition may further comprise another agent, including, for example, a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. In some embodiments the formulation includes an alkylating agent (e.g., chlorambucil, bendamustine hydrochloride or cyclophosphamide) a nucleoside analog (e.g., fludurabine, pentostatin, cladribine or cytarabine) a corticosteroid (e.g., prednisone, prednisolone or methylprednisolone), an immunomodulatory agent (e.g., lenalidomide), an antibiotic (e.g., doxorubicin, daunorubicin idarubicin or mitoxentrone), a synthetic flavon (e.g., flavopiridol), a Bcl2 antagonist, (e.g., oblimersen or ABT-263), a hypomethylating agent (e.g., azacytidine or decitabine), an FLT3 inhibitor (e.g., midostaurin, sorafenib and AC220). Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

The antibodies of the present disclosure may be formulated in any suitable form for delivery to a target cell/tissue, for example, as microcapsules or macroemulsions (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980); Park et al., Molecules 10: 146-161 (2005); Malik et al., Curr. Drug. Deliv. 4: 141-151 (2007)); as sustained release formulations (Putney and Burke, Nature Biotechnol. 16: 153-157, (1998)) or in liposomes (Maclean et al., Int. J. Oncol. 11: 235-332 (1997); Kontermann, Curr. Opin. Mol. Ther. 8: 39-45 (2006)).

An antibody provided herein can also be entrapped in microcapsule prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and poly-(methylmethacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed, for example, in Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.

Various delivery systems are known and can be used to administer a prophylactic or therapeutic agent (e.g., an antibody that binds to GFRAL as described herein), including, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. In another embodiment, a prophylactic or therapeutic agent, or a composition provided herein can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see, e.g., Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent (e.g., an antibody that binds to GFRAL as described herein) or a composition of the invention (see, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable.

In yet another embodiment, a controlled or sustained release system can be placed in proximity of the therapeutic target, for example, the nasal passages or lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Controlled release systems are discussed, for example, by Langer (1990, Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more antibodies that bind to GFRAL as described herein. (See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al., 1996, “Intratumoral Radioimmunotherapy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, “Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760).

Additional delivery systems can be used to administer a prophylactic or therapeutic agent (e.g., an antibody that binds to GFRAL as described herein) including, but not limited to, injectable drug delivery devices. Injectable drug delivery devices for anti-GFRAL antibodies include, for example, hand-held devices or wearable devices. Hand-held devices useful for anti-GFRAL antibodies include autoinjectors, such as the FLEXIQ-DV (Elcam Medical) or PRO-JECT (Aptar Pharma) autoinjectors. Wearable devices useful for anti-GFRAL antibodies include, for example, on-body drug delivery systems, such as the NEULASTA drug delivery kit (Amgen). Injectable drug delivery devices for anti-GFRAL antibodies can contain the antibody as a prophyactic or therapeutic agent, for example, in prefilled syringes, cartridges or vials. Injectable drug delivery devices (e.g., autoinjectors) and/or their containers (e.g., syringes, cartridges or vials) for anti-GFRAL antibodies can be disposable. Exemplary injectable devices useful for anti-GFRAL antibodies are described in WO2014/081780.

In some embodiments, additional drug delivery systems can be used to administer a prophylactic or therapeutic agent (e.g., an antibody that binds to GFRAL as described herein) including, but not limited to, osmotic pumps. Different types of osmotic pump systems for anti-GFRAL antibodies can be used, including, for example, single compartment systems, dual compartment systems, and multiple compartment systems. Exemplary osmotic pump systems useful for anti-GFRAL antibodies are described, for example, in Herrlich et al. (2012) Advanced Drug Delivery Reviews 64, 1617-1627. In some embodiments, the osmotic pump for anti-GFRAL antibodies can include implantable drug-dispensing osmotic pumps such as the DUROS pump.

Therapeutic Methods

An anti-GFRAL antibody of the present disclosure may be used in, for example, therapeutic methods.

In some embodiments, the present disclosure provides methods for treating or preventing a GDF15-mediated disease, disorder, or condition, wherein the method comprises administering an anti-GFRAL antibody or fragment thereof described herein to a subject suffering from a GDF15-mediated disease, disorder or condition. Additionally, the subject can be administered a pharmaceutical composition comprising an anti-GFRAL antibody or fragment thereof described herein.

In some embodiments, present disclosure provides a method to treat a subject suffering from involuntary weight loss. An example of a suitable subject may be one who is diagnosed with a wasting disease or cachexia. Suitable patients include those suffering from liver cirrhosis, hyperthyroidism, chronic kidney disease, Parkinson's disease, cancer, eating disorder (e.g., anorexia nervosa), chronic inflammatory disease (e.g., rheumatoid arthritis), sepsis or other forms of systemic inflammation, chronic obstructive pulmonary disease, AIDS, tuberculosis, and muscle wasting, such as muscular dystrophy or multiple sclerosis), or sarcopenia.

In some embodiments, the present disclosure also provides methods for preventing involuntary weight loss in a subject who may be at risk of involuntary weight loss due to a chronic disease, such as, liver cirrhosis, hyperthyroidism, chronic kidney disease, Parkinson's disease, cancer, eating disorder (e.g., anorexia nervosa), chronic inflammatory disease (e.g., rheumatoid arthritis), sepsis or other forms of systemic inflammation, chronic obstructive pulmonary disease, AIDS, tuberculosis, and muscle wasting, such as muscular dystrophy or multiple sclerosis), or sarcopenia. Such subjects may include subjects who have elevated levels of GDF15, are undergoing treatment for cancer, and the like.

In some embodiments, the present disclosure provides a method to treat a subject suffering from cachexia. An example of a suitable subject is one who is diagnosed with cachexia. The present disclosure also provides methods for preventing involuntary weight loss in a subject who may be at risk of involuntary weight loss due to onset of cachexia. Such subjects include subjects who have elevated levels of GDF15, have cancer, are undergoing treatment for cancer, have an eating disorder, and the like.

Also disclosed is a method for modulating GDF15 activity in a patient having elevated GDF15 activity. As used herein, “elevated GDF15 activity” refers to increased activity or amount of GDF15 in a biological fluid of a subject in comparison to a normal subject. A number of conditions are associated with increased GDF15 serum level, wherein the increased GDF15 results in a number of symptoms such as appetite loss, weight loss, and the like. Examples of conditions associated with increased GDF15 serum level include cancer, e.g., melanoma, gastric cancer, pancreatic cancer, prostate cancer; autoimmune diseases such as, arthritis and inflammation; cardiovascular diseases like atherosclerosis, heart failure, hypertension, myocardial infarction, chest pain, and cardiovascular events; metabolic diseases like anemia, cachexia, anorexia, kidney disease, and thalassemia, etc.

A subject having any of the above diseases, disorders or conditions is a suitable candidate for receiving an anti-GFRAL antibody or fragment thereof described herein, or a combination of a therapeutic agent and the anti-GFRAL antibody or fragment thereof.

Administering the subject an anti-GFRAL antibody or fragment thereof to such a subject can decrease or prevent one or more of the symptoms associated with a GDF15-mediated disease, disorder or condition. For example, administering an anti-GFRAL antibody or fragment thereof of the present disclosure can increase body weight and/or appetite in a subject. As another example, administering an anti-GFRAL antibody or fragment thereof of the present disclosure can maintain body weight in a subject or reduce body weight loss is a subject.

In one aspect, methods are provided for treating a disease, disorder or condition comprising administering to an individual an effective amount of an anti-GFRAL antibody or fragment thereof. In certain embodiments, a method for treating a disease, disorder, or condition comprises administering to an individual an effective amount of a pharmaceutical formulation comprising an anti-GFRAL antibody and, optionally, at least one additional therapeutic agent, such as those described herein.

An anti-GFRAL antibody or fragment thereof can be administered to a human for therapeutic purposes. Moreover, an anti-GFRAL antibody or fragment thereof can be administered to a non-human mammal expressing GFRAL with which the antibody cross-reacts (e.g., a primate, pig, rat, or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the present disclosure (e.g., testing of dosages and time courses of administration).

Antibodies of the present disclosure can be used either alone or in combination with other compositions in a therapy. For example, an anti-GFRAL antibody of the present disclosure may be co-administered with at least one additional therapeutic agent and/or adjuvant. In some embodiments, the additional compound is a therapeutic antibody other than an anti-GFRAL antibody.

Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of an anti-GFRAL antibody or fragment thereof of the present disclosure can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant. Antibodies of the present disclosure can also be used in combination with additional therapeutic regimens including, without limitation, those described herein.

An anti-GFRAL antibody of the present disclosure (and any additional therapeutic agent or adjuvant) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody or conjugate is suitably administered by pulse infusion, particularly with declining doses of the antibody or fragment thereof. Dosing can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

Antibodies of the present disclosure would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The anti-GFRAL antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody or immunoconjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of a disease, disorder, or condition, the appropriate dosage of an anti-GFRAL antibody or fragment thereof of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents, such as agents described herein) will depend on the type of disease, disorder, or condition, to be treated, the type of antibody, the severity and course of the disease, disorder, or condition, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the antibody, and the discretion of the attending physician. The anti-GFRAL antibody or fragment thereof is suitably administered to the subject at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 100 mg/kg (e.g., 0.1 mg/kg-20 mg/kg, 1 mg/kg-15 mg/kg, etc.) of antibody or fragment thereof can be an initial candidate dosage for administration to the subject, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. Exemplary dosages of the antibody or fragment thereof may be in the range from about 0.05 mg/kg to about 10.0 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg, or 10.0 mg/kg (or any combination thereof) of antibody may be administered to the subject. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the subject receives from about two to about twenty, or e.g., about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. An exemplary dosing regimen comprises administering an initial loading dose, followed by a maintenance dose (e.g., weekly) of the antibody or fragment thereof. The initial loading dose may be greater than the maintenance dose. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

In some embodiments, the method described herein involves administering the subject an anti-GFRAL antibody or fragment thereof to a patient who has involuntary body weight loss or is at risk of developing involuntary body weight loss. The subject methods include administering an anti-GFRAL antibody or fragment thereof disclosed herein to a subject who has elevated serum levels of GDF15. In certain embodiment, the antibody or fragment thereof is an anti-GFRAL antibody that competes with GDF15 for binding to extracellular domain of GFRAL. In certain embodiments, the antibody or fragment thereof binds to an extracellular domain of a GFRAL protein but does not activate RET. For example, the antibody or fragment thereof is an anti-GFRAL antibody that competes with GDF15 for binding to extracellular domain of GFRAL but does not activate RET upon binding to GFRAL. Such an antibody is described herein.

In the methods of the present disclosure, anti-GFRAL antibodies or fragments thereof described herein or pharmaceutical compositions comprising said antibodies or fragments thereof can be administered to a subject (e.g., a human patient) to, for example, achieve a target body weight and/or maintain body weight; achieve a target body mass index (BMI) and/or maintain a BMI; increase appetite; and the like. A normal human adult has a BMI in the range 18.5-24.9 Kg/m². The subject treatment methods can increase body weight, BMI, muscle weight, and/or food intake in a patient by at least about 5%, e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.

The methods relating to treatment or prevention of a GDF15-mediated disease, disorder or condition (e.g., involuntary weight loss) described herein include, for example, use of an anti-GFRAL antibody or fragment thereof described herein for therapy/prevention alone or in combination with other types of therapy. The method involves administering to a subject the anti-GFRAL antibody or fragment thereof and another agent.

In some embodiments, the agent is administered to a patient experiencing loss of muscle mass, for example, loss of muscle mass associated with an underlying disease. Underlying diseases associated with cachexia include, but are not limited to, cancer, chronic renal disease, chronic obstructive pulmonary disease, AIDS, tuberculosis, chronic inflammatory diseases, sepsis and other forms of systemic inflammation, muscle wasting, such as muscular dystrophy, and the eating disorder known as anorexia nervosa. In some embodiments, the agent inhibits loss of lean mass (e.g., muscle mass) and or fat mass by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%.

In some embodiments, a loss of lean mass (e.g., muscle mass) is accompanied by a loss of fat mass. In some embodiments, the agent can inhibit loss of fat mass by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%.

In some embodiments, the agent is administered to a patient diagnosed with body weight loss (e.g., involuntary weight loss). In some embodiments, the agent can revert body weight loss (e.g., involuntary weight loss) by at least 2%, 5%, 10%, 15%, 20%, 25%, 30% or 35%.

In some embodiments, the agent is administered to a patient diagnosed with loss of organ mass, for example, loss of organ mass associated with an underlying disease. Underlying diseases associated with cachexia include, but are not limited to, cancer, chronic renal disease, chronic obstructive pulmonary disease, AIDS, tuberculosis, chronic inflammatory diseases, sepsis and other forms of systemic inflammation, muscle wasting, such as muscular dystrophy, and the eating disorder known as anorexia nervosa. In some embodiments, the agent can inhibit loss of organ mass by at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%. In some embodiments, loss of organ mass is observed in heart, liver, kidney, and/or spleen. In some embodiments, the loss of organ mass in accompanied by a loss of muscle mass, a loss of fat mass and/or involuntary weight loss.

Sarcopenia, muscle wasting disorders and significant muscle weight loss can occur in the absence of cachexia, decreased appetite or body weight loss. In some embodiments, the agent can be used to treat a subject diagnosed with sarcopenia, a muscle wasting disorder and/or significant muscle weight loss, whether or not the subject has, or has been diagnosed with, cachexia or decreased appetite. Such a method comprises administering a therapeutically effective amount of one or more agents to a subject in need thereof.

Any of a wide variety of therapies directed to treating or preventing cachexia can be combined in a composition or therapeutic method with the subject proteins.

Where the GFRAL-ECD protein is administered in combination with one or more other therapies, the combination can be administered anywhere from simultaneously to up to 5 hours or more, e.g., 10 hours, 15 hours, 20 hours or more, prior to or after administration of a subject protein. In certain embodiments, a subject protein and other therapeutic intervention are administered or applied sequentially, e.g., where a subject protein is administered before or after another therapeutic treatment. In yet other embodiments, a subject protein and other therapy are administered simultaneously, e.g., where a subject protein and a second therapy are administered at the same time, e.g., when the second therapy is a drug it can be administered along with a subject protein as two separate formulations or combined into a single composition that is administered to the subject. Regardless of whether administered sequentially or simultaneously, as illustrated above, the treatments are considered to be administered together or in combination for purposes of the present disclosure.

Cytokines that are implicated in cachexia include Activin A and IL-6. Increased activin levels have been associated with cancer-associated cachexia and gonadal tumors. See, e.g., Marino et al. (2013) CYTOKINE & GROWTH FACTOR REV. 24:477-484. Activin A is a member of the TGF-beta family, and is a ligand of the activin type 2 receptor, ActRIIB. See, e.g., Zhou et al. (2010) CELL 142:531-543. Circulating levels of IL-6 have been shown to correlate with weight loss in cancer patients, as well as with reduced survival. See, e.g., Fearon et al. (2012) CELL METABOLISM 16: 153-166.

Accordingly, in some embodiments, one or more inhibitors of Activin-A or the Activin-A receptor, ActRIIB, IL-6 or the IL-6 receptor (IL-6R), can be administered in combination with (for example, administered at the same time as, administered before, or administered after) an anti-GFRAL antibody or fragment thereof described herein. Exemplary inhibitors of Activin A or ActRIIB, include, for example, an anti-Activin-A antibody or an antigen binding fragment thereof, an anti-ActRIIB antibody or an antigen binding fragment thereof, a small molecule inhibitor of Activin-A, a small molecule inhibitor of ActRIIB, and a ‘decoy’ receptor of ActRIIB, such as a soluble ActRIIB receptor and a fusion of the soluble ActRIIB receptor with an Fc molecule (ActRIIB-Fc). See, e.g., Zhou et al. (2010), supra. Suitable inhibitors of IL-6 or IL-6R, include an anti-IL-6 antibody or an antigen binding fragment thereof, an anti-IL-6R antibody or an antigen binding fragment thereof, a small molecule inhibitor of IL-6, a small molecule inhibitor of IL-6R, and a ‘decoy’ receptor of IL-6R, such as a soluble IL-6 receptor and a fusion of the soluble IL-6 receptor with an Fc molecule (IL6R-Fc). See, e.g., Enomoto et al. (2004) BIOCHEM. AND BIOPHYS. RES. COMM. 323: 1096-1 102; Argiles et al. (2011) EUR. J. PHARMACOL. 668:S81-S86; Tuca et al. (2013) ONCOLOGY/HEMATOLOGY 88:625-636. Suitable inhibitors of IL-6 or IL-6R can include, e.g., Tocilizumab (Actemra®, Hoffmann-LaRoche), a humanized anti-IL-6R monoclonal antibody approved for treatment of rheumatoid arthritis, and Sarilumab/REGN88 (Regeneron), a humanized anti-IL6R antibody in clinical development for treatment of rheumatoid arthritis; and Selumetinib/AZD6244 (AstraZeneca), an allosteric inhibitor of MEK, which has been shown to inhibit IL-6 production. Prado et al. (2012) BRITISH J. CANCER 106: 1583-1586.

TNFα and IL-1 are cytokines known to be involved in mediation of the proinflammatory response, which are also implicated in muscle depletion, anorexia and cachexia. Increased circulating levels of TNFα appear to inhibit myogenesis. TNFα, also known as “cachectin,” stimulates interleukin-1 secretion and is implicated in the induction of cachexia. IL-1 is a potent trigger of the acute-phase inflammatory response, and it has been shown that infusion of IL-1 can lead to marked weight loss and appetite loss. IL-1 has been shown to contribute to the initiation of cancer cachexia in mice bearing a murine colon-26 adenocarcinoma (Strassmann et al. (1993) J. IMMUNOL. 150:2341). See also, Mathys and Billiau (1997) NUTRITION 13:763-770; Fong et al. (1989) AM. J. PHYSIOL.—REGULATORY, INTEGRATIVE AND COMPARATIVE PHYSIOL., 256:R659-R665. Thus, TNFα inhibitors and IL-1 inhibitors that are used in the treatment of rheumatoid arthritis may also be useful in the treatment of cachexia.

Accordingly, in some embodiments, one or more inhibitors of TNFα or IL-1 can be administered in combination with (e.g., administered at the same time as, administered before, or administered after) an anti-GFRAL antibody or fragment thereof described herein. Suitable inhibitors of TNFα or IL-1 include an anti-TNFα antibody or an antigen binding fragment thereof, an anti-IL-1 antibody or an antigen binding fragment thereof, a small molecule inhibitor of TNFα or IL-1, and a ‘decoy’ receptor of TNFα or IL-1, such as a soluble TNFα or IL-1 receptor and a fusion of the soluble form of TNFα or IL-1 with an Fc molecule. Suitable inhibitors of TNFα include for example, etanercept (Enbrel®, Pfizer/Amgen), infliximab (Remicade®, Janssen Biotech), adalimumab (Humira®, Abbvie), golimumab (Simponi®, Johnson and Johnson/Merck), and certolizumab pegol (Cimzia®, UCB). Suitable IL-1 inhibitors include, e.g., Xilonix® antibody that targets IL-1 a (XBiotech), anikinra (Kinaret®, Amgen), canakinumab (Ilaris®, Novartis), and rilonacept (Arcalyst®, Regeneron). In certain embodiments, the TNFα inhibitor or IL-1 inhibitor, which is typically administered systemically for the treatment of rheumatoid arthritis may be administered locally and directly to the tumor site.

Myostatin, also known as GDF-8, is a member of the TGF-3 family of peptides that is a negative regulator of muscle mass, as shown by increased muscle mass in myostatin deficient mammals. Myostatin is a ligand of the activin type 2 receptor, ActRIIB.

Accordingly, in some embodiments, one or more inhibitors of myostatin or its receptor may be administered in combination with (for example, administered at the same time as, administered before, or administered after) an anti-GFRAL antibody or fragment thereof described herein. Suitable inhibitors of myostatin or ActRIIB, include an anti-myostatin antibody or an antigen binding fragment thereof, an anti-ActRIIB antibody or an antigen binding fragment thereof, a small molecule inhibitor of myostatin, a small molecule inhibitor of ActRIIB, and a ‘decoy’ receptor of GDF-8, such as a soluble ActRIIB and a fusion of the soluble form of ActRIIB with an Fc molecule. See, e.g., Lokireddy et al. (2012) BIOCHEM. J. 446(I):23-26. Myostatin inhibitors that may be suitable for the present methods include REGN1033 (Regeneron); see Bauerlein et al. (2013) J. CACHEXIA SARCOPENIA MUSCLE: Abstracts of the 7th Cachexia Conference, Kobe/Osaka, Japan, Dec. 9-11, 2013, Abstract 4-06; LY2495655 (Lilly), a humanized anti-myostatin antibody in clinical development by Eli Lilly; see also “A PHASE 2 STUDY OF LY2495655 IN PARTICIPANTS WITH PANCREATIC CANCER,” available on the world wide web at clinicaltrials.gov/ct2/NCT01505530; NML identifier: NCT01505530; ACE-031 (Acceleron Pharma); and stamulumab (Pfizer).

Agents such as Ghrelin or ghrelin mimetics, or other growth hormone secretagogues (GHS) which are able to activate the GHS receptor (GHS-RIa), also known as the ghrelin receptor, can be useful for increasing food intake and body weight in humans. See Guillory et al. (2013) in VITAMINS AND HORMONES vol. 92, chap. 3; and Steinman and DeBoer (2013) VITAMINS AND HORMONES vol. 92, chap. 8. Accordingly, in some embodiments, one or more Ghrelin or ghrelin mimetics, or other growth hormone secretagogues (GHS), can be administered in combination with (for example, administered at the same time as, administered before, or administered after) an anti-GFRAL antibody or fragment thereof described herein. Suitable ghrelin mimetics include anamorelin (Helsinn, Lugano, CH); see Temel et al. (2013) J. CACHEXIA SARCOPENIA MUSCLE: Abstracts of the 7th Cachexia Conference, Kobe/Osaka, Japan, Dec. 9-11, 2013, Abstract 5-01. Other suitable GHS molecules can be identified, for example, using the growth hormone secretagogue receptor Ghrelin competition assay described in PCT Publication Nos. WO201 1/1 17254 and WO2012/113103.

Agonists of the androgen receptor, including small molecules and other selective androgen receptor modulators (SARMs) can be useful in treating cachexia and/or sarcopenia. See, e.g., Mohler et al. (2009) J. MED. CHEM. 52:3597-3617; Nagata et al. (2011) BIOORGANIC AND MED. CHEM. LETTERS 21: 1744-1747; and Chen et al. (2005) MOL. INTERV. 5: 173-188. Ideally, SARMs should act as full agonists, like testosterone, in anabolic target tissues, such as muscle and bone, but should demonstrate only partial or pure androgen receptor antagonistic activities on prostate tissue. See, e.g., Bovee et al. (2010) J. STEROID BIOCHEM. & MOL. BIOL. 118:85-92. Suitable SARMs can be identified, e.g., by use of the methods and assays described in Zhang et al. (2006) BIOORG. MED. CHEM. LETT. 16:5763-5766; and Zhang et al. (2007) BIOORG. MED. CHEM. LETT. 17:439-443.

Accordingly, in some embodiments, one or more androgen receptor agonists can be administered in combination with (for example, administered at the same time as, administered before, or administered after) an anti-GFRAL antibody or fragment thereof described herein. Suitable SARMs include, for example, GTx-024 (enobosarm, Ostarine®, GTx, Inc.), a SARM in phase II clinical development by GTx, Inc. See also, Dalton et al. (2011) J. CACHEXIA SARCOPENIA MUSCLE 2: 153-161. Other suitable SARMs include 2-(2,2,2)-trifluoroethyl-benzimidazoles (Ng et al. (2007) BIOORG. MED. CHEM. LETT. 17: 1784-1787) and JNJ-26146900 (Allan et al. (2007) J. STEROID BIOCHEM. & MOL. BIOL. 103:76-83).

β-adrenergic receptor blockers, or beta-blockers, have been studied for their effect on body weight in cachexia subjects, and have been associated with partial reversal of cachexia in patients with congestive heart failure. See, e.g., Hryniewicz et al. (2003) J. CARDIAC FAILURE 9:464-468. Beta-blocker MT-102 (PsiOxus Therapeutics, Ltd.) has been evaluated in a phase 2 clinical trial for subjects with cancer cachexia. See Coats et al. (2011) J. CACHEXIA SARCOPENIA MUSCLE 2:201-207. Accordingly, in some embodiments, one or more β-adrenergic receptor blockers, or beta-blockers, can be administered in combination with (for example, administered at the same time as, administered before, or administered after) an anti-GFRAL antibody or fragment thereof described herein.

Melanocortin receptor-knockout mice with a genetic defect in melanocortin signaling exhibit a phenotype opposite that of cachexia: increased appetite, increased lean body mass, and decreased metabolism. Thus, melanocortin antagonism has emerged as a potential treatment for cachexia associated with chronic disease (DeBoer and Marks (2006) TRENDS IN ENDOCRINOLOGY AND METABOLISM 17: 199-204).

Accordingly, in some embodiments, one or more inhibitors of a melanocortin peptide or a melanocortin receptor can be administered in combination (e.g., administered at the same time as, administered before, or administered after) with an anti-GFRAL antibody or fragment thereof described herein. Suitable inhibitors of melanocortins or melanocortin receptors include an anti-melanocortin peptide antibody or an antigen binding fragment thereof, an anti-melanocortin receptor antibody or an antigen binding fragment thereof, a small molecule inhibitor of a melanocortin peptide, a small molecule inhibitor of a melanocortin receptor, and a ‘decoy’ receptor of a melanocortin receptor, such as soluble melanocortin receptor and a fusion of a soluble melanocortin receptor with an Fc molecule. Suitable melacortin receptor inhibitors include, for example, the melanocortin receptor antagonist agouri-related peptide (AgRP(83-132)), which has been demonstrated to prevent cachexia-related symptoms in a mouse model of cancer-related cachexia (Joppa et al. (2007) PEPTIDES 28:636-642).

Anti-cancer agents, especially those that can cause cachexia and elevate GDF15 levels, such as cisplatin, can be used in methods of the present disclosure in combination with (for example, administered at the same time as, administered before, or administered after) an anti-GFRAL antibody or fragment thereof described herein. Many cancer patients are weakened by harsh courses of radio- and/or chemotherapy, which can limit the ability of the patient to tolerate such therapies, and hence restrict the dosage regimen. Certain cancer agents themselves, such as fluorouracil, adriamycin, methotrexate and cisplatin, can contribute to cachexia, for example by inducing severe gastrointestinal complications. See, e.g., Inui (2002) CANCER J. FOR CLINICIANS 52:72-91. By the methods of the present disclosure, in which an anti-cancer agent is administered in combination with an anti-GFRAL antibody of the disclosure, it is possible to decrease the incidence and/or severity of cachexia, and ultimately increase the maximum tolerated dose of such an anti-cancer agent. Accordingly, efficacy of treatment with anti-cancer agents that can cause cachexia can be improved by reducing the incidence of cachexia as a dose-limiting adverse effect, and by allowing administration of higher doses of a given anticancer agent.

Thus, provided herein are pharmaceutical compositions comprising an anti-GFRAL antibody or fragment thereof described herein in combination with an agent selected from the group consisting of an inhibitor of Activin-A, an inhibitor of ActRIIB, an inhibitor of IL-6 or an inhibitor of IL-6R, a ghrelin, a ghrelin mimetic or a GHS-RIa agonist, a SARM, a TNFα inhibitor, an IL-Iα inhibitor, a myostatin inhibitor, a beta-blocker, a melanocortin peptide inhibitor, a melanocortin receptor inhibitor, and an anti-cancer agent. The present disclosure also includes methods of treating, preventing or minimizing cachexia and/or sarcopenia in a mammal comprising administering to a mammal in need thereof a pharmaceutical composition or compositions comprising an effective amount of an anti-GFRAL antibody of the disclosure in combination with an effective amount of an inhibitor of Activin-A, an inhibitor of ActRIIB, an inhibitor of IL-6 or an inhibitor of IL-6R, a ghrelin, a ghrelin mimetic or a GHS-RIa agonist, a SARM, a TNFα inhibitor, an IL-Iα inhibitor, a myostatin inhibitor, a beta-blocker, a melanocortin peptide inhibitor, or a melanocortin receptor inhibitor.

In another aspect, provided herein is a method of inhibiting loss of muscle mass associated with an underlying disease comprising administering to a mammal in need thereof a pharmaceutical composition or compositions comprising an effective amount of an anti-GFRAL antibody or fragment thereof described herein in combination with an effective amount of an inhibitor of Activin-A, an inhibitor of ActRIIB, an inhibitor of IL-6 or an inhibitor of IL-6R, a ghrelin, a ghrelin mimetic or a GHS-RIa agonist, a SARM, a TNFα inhibitor, an IL-Iα inhibitor, a myostatin inhibitor, a beta-blocker, a melanocortin peptide inhibitor, or a melanocortin receptor inhibitor to prevent or reduce loss of muscle mass. The underlying disease can be selected from the group consisting of cancer, chronic heart failure, chronic kidney disease, chronic obstructive pulminary disease, AIDS, multiple sclerosis, rheumatoid arthritis, sepsis, and tuberculosis. Additionally, in some embodiments, the loss of muscle mass is accompanied by a loss of fat mass.

In another aspect, provided herein is a method of inhibiting or reducing involuntary weight loss in a mammal comprising administering to a mammal in need thereof a pharmaceutical composition or pharmaceutical compositions comprising an effective amount of an anti-GFRAL antibody of the disclosure in combination with an effective amount of an inhibitor of Activin-A, an inhibitor of ActRIIB, an inhibitor of IL-6 or an inhibitor of IL-6R, a ghrelin, a ghrelin mimetic or a GHS-RIa agonist, a SARM, a TNFα inhibitor, a IL-Iα inhibitor, a myostatin inhibitor, a beta-blocker, a melanocortin peptide inhibitor, or a melanocortin receptor inhibitor.

Certain anti-cancer agents, such as cisplatin, have one or more undesirable adverse effects that involve causing or increasing one or more syndromes such as cachexia, sarcopenia, muscle wasting, bone wasting or involuntary body weight loss. Accordingly, in another aspect, provided herein is a method of treating cancer, while preventing, minimizing or reducing the occurrence, frequency or severity of cachexia, sarcopenia, or muscle wasting, bone wasting or involuntary loss of body weight in a mammal, comprising administering to a mammal in need thereof a pharmaceutical composition comprising an effective amount of an anti-GFRAL antibody or fragment thereof described herein in combination with one or more anti-cancer agents. In some embodiments, the method of treating cancer, while preventing, minimizing or reducing the occurrence, frequency or severity of cachexia, sarcopenia or muscle wasting, bone wasting or involuntary loss of body weight in a mammal, comprises administering to a mammal in need thereof a pharmaceutical composition comprising an effective amount of an anti-GFRAL antibody or fragment thereof described herein in combination with one or more anti-cancer agents known to cause or increase the occurrence, frequency or severity of cachexia, sarcopenia, or muscle wasting, bone wasting or involuntary loss of body weight in a mammal.

Diagnostic Methods and Methods of Detection

In one aspect, anti-GFRAL antibodies and fragments thereof of the present disclosure are useful for detecting the presence of GFRAL in a biological sample. Such anti-GFRAL antibodies can include those that bind to human GFRAL. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain embodiments, a biological sample comprises a cell or tissue.

In one aspect, the present disclosure provides a method of detecting the presence of GFRAL in a biological sample. In certain embodiments, the method comprises contacting the biological sample with an anti-GFRAL antibody under conditions permissive for binding of the anti-GFRAL antibody to GFRAL, and detecting whether a complex is formed between the anti-GFRAL antibody and GFRAL.

In one aspect, the present disclosure provides a method of diagnosing a disorder associated with expression of GFRAL. In certain embodiments, the method comprises contacting a test cell with an anti-GFRAL antibody; determining the level of expression (either quantitatively or qualitatively) of GFRAL by the test cell by detecting binding of the anti-GFRAL antibody to GFRAL; and comparing the level of expression of GFRAL by the test cell with the level of expression of GFRAL by a control cell (e.g., a normal cell of the same tissue origin as the test cell or a cell that expresses GFRAL at levels comparable to such a normal cell), wherein a higher level of expression of GFRAL by the test cell as compared to the control cell indicates the presence of a disorder associated with increased expression of GFRAL. In certain embodiments, the test cell is obtained from an individual suspected of having a disease, disorder or condition associated with expression of GDF15 and/or a disease, disorder or condition in which it is desirable to inhibit the in vivo effects of GDF15. In certain embodiments, the disease, disorder or condition is, for example, involuntary weight loss. Such exemplary diseases, disorders or conditions may be diagnosed using an anti-GFRAL antibody of the present disclosure.

In certain embodiments, a method of diagnosis or detection, such as those described above, comprises detecting binding of an anti-GFRAL antibody to GFRAL expressed on the surface of a cell or in a membrane preparation obtained from a cell expressing GFRAL on its surface. In certain embodiments, the method comprises contacting a cell with an anti-GFRAL antibody under conditions permissive for binding of the anti-GFRAL antibody to GFRAL, and detecting whether a complex is formed between the anti-GFRAL antibody and GFRAL on the cell surface. An exemplary assay for detecting binding of an anti-GFRAL antibody to GFRAL expressed GFRAL on the surface of a cell is a “FACS” assay.

Certain other methods can be used to detect binding of anti-GFRAL antibodies to GFRAL. Such methods include, but are not limited to, antigen-binding assays that are well known in the art, such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, fluorescent immunoassays, protein A immunoassays, and immunohistochemistry (IHC).

In certain embodiments, anti-GFRAL antibodies are labeled. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, for example, through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, for example, firefly luciferase and bacterial luciferase (see, e.g., U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

In certain embodiments, anti-GFRAL antibodies are immobilized on an insoluble matrix. Immobilization entails separating the anti-GFRAL antibody from any GFRAL that remains free in solution. This conventionally is accomplished by either insolubilizing the anti-GFRAL antibody before the assay procedure, as by adsorption to a water-insoluble matrix or surface (see, e.g., Bennich et al., U.S. Pat. No. 3,720,760), or by covalent coupling (for example, using glutaraldehyde cross-linking), or by insolubilizing the anti-GFRAL antibody after formation of a complex between the anti-GFRAL antibody and GFRAL, for example, by immunoprecipitation.

Any of the above embodiments of diagnosis or detection may be carried out using an immunoconjugate of the present disclosure in place of or in addition to an anti-GFRAL antibody.

Assays

Anti-GFRAL antibodies of the present disclosure may be characterized for their physical/chemical properties and/or biological activities by various assays known in the art.

1. Activity Assays

In one aspect, assays are provided for identifying anti-GFRAL antibodies thereof having biological activity. Biological activity can include, for example, assays which measure effects on glucose and/or lipid metabolism. For example, a blood glucose assay can be used. Blood glucose (e.g., in mouse tail snip or in a human blood sample) can be measured using ACCU-CHEK Active test strips read by ACCU-CHEK Active meter (Roche Diagnostics, Indianapolis, Ind.) following manufacturer's instruction. In addition, for example, a lipid profile assay can be used. Whole blood (e.g., from mouse tail snips or from a human blood sample) can be collected into plain capillary tubes (BD Clay Adams SurePrep, Becton Dickenson and Co. Sparks, Md.). Serum and blood cells can be separated by spinning the tubes in an Autocrit Ultra 3 (Becton Dickinson and Co. Sparks, Md.). Serum samples can be assayed for lipid profile (triglyceride, total cholesterol, HDL, and non-HDL) using Integra 400 Clinical Analyzer (Roche Diagnostics, Indianapolis, Ind.) following the manufacturer's instructions.

2. Binding Assays and Other Assays

In one aspect, an anti-GFRAL antibody is tested for its antigen binding activity. For example, in certain embodiments, an anti-GFRAL antibody is tested for its ability to bind to exogenous or endogenous GFRAL expressed on the surface of a cell. A FACS assay may be used for such testing.

A panel of monoclonal antibodies raised against GFRAL may be grouped based upon the epitiopes they recognize, a process known as epitope binning. Epitope binning is typically carried out using competition assays, which evaluate an antibody's ability to bind to an antigen in the presence of another antibody. In an exemplary competition assay, immobilized GFRAL is incubated in a solution comprising a first labeled antibody that binds to GFRAL and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to GFRAL. The second antibody may be present in a hybridoma supernatant. As a control, immobilized GFRAL is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to GFRAL, excess unbound antibody is removed, and the amount of label associated with immobilized GFRAL is measured. If the amount of label associated with immobilized GFRAL is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to GFRAL. In certain embodiments, immobilized GFRAL is present on the surface of a cell or in a membrane preparation obtained from a cell expressing GFRAL on its surface.

High-throughput methods of epitope binning are also known in the art (see, e.g., Jia et al., J. Immunol. Methods 2004, 288(1-2):91-98, describing a method of multiplexed competitive antibody binning for the characterization of monoclonal antibodies; and Miller et al., J. Immunol. Methods 2011, 365(1-2):118-25, describing epitope binning of murine monoclonal antibodies by a multiplexed pairing assay).

3. Epitope Mapping

Epitope mapping is the process of identifying the binding sites, or epitopes, of an antibody on its target protein antigen (e.g., epitopes of an anti-GFRAL antibody on GFRAL). Antibody epitopes may be linear epitopes or conformational epitopes. Linear epitopes are formed by a continuous sequence of amino acids in a protein. Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three-dimensional structure.

A variety of methods are known in the art for mapping antibody epitopes on target protein antigens. These include mutagenesis methods, peptide scanning methods, display methods, methods involving and mass spectroscopy, and structural determination.

The site directed mutagenesis method involves targeted site-directed mutagenesis where critical amino acids are identified by systematically introducing substitutions along the protein sequence and then determining the effects of each substitution on antibody binding. This may be done by “alanine scanning mutagenesis,” as described, for example, by Cunningham and Wells (1989) Science 244: 1081-1085, or some other form of point mutagenesis of amino acid residues in human GFRAL. Mutagenesis studies, however, may also reveal amino acid residues that are crucial to the overall three-dimensional structure of GFRAL but that are not directly involved in antibody-antigen contacts, and thus other methods may be necessary to confirm a functional epitope determined using this method.

Shotgun mutagenesis mapping utilizes a comprehensive plasmid-mutation library for the target gene, with each clone in the library bearing a unique amino acid mutation and the entire library covering every amino acid in the target protein. The clones that constitute the mutation library are individually arranged in microplates, expressed within living mammalian cells, and tested for immunoreactivity with antibodies of interest. Amino acids critical for antibody epitopes are identified by a loss of reactivity and are then mapped onto a protein structure to visualize epitopes. By automating the analysis, new epitope maps can be derived within days to weeks. Because it uses the native structure of proteins within mammalian cells, the technique allows both linear and conformational epitope structures to be mapped on complex proteins. (See, e.g., Paes et al., J. Am. Chem. Soc. 131(20): 6952-6954 (2009); Banik and Doranz, Genetic Engineering and Biotechnology News 3(2): 25-28 (2010)).

The epitope bound by an anti-GFRAL antibody may also be determined using peptide scanning methods. In peptide scanning, libraries of short peptide sequences from overlapping segments of the target protein, GFRAL, are tested for their ability to bind antibodies of interest. The peptides are synthesized and screened for binding, e.g., using ELISA or BIACORE, or on a chip, by any of the multiple methods for solid-phase screening (see, e.g., Reineke et al., Curr. Opin. Biotechnol. 12: 59-64, 2001) as in the “pepscan” methodology (see, e.g., WO 84/03564; WO 93/09872). Such peptide screening methods may not be capable of detecting some discontinuous functional epitopes, i.e. functional epitopes that involve amino acid residues that are not contiguous along the primary sequence of the GFRAL polypeptide chain.

A recently developed technology termed CLIPS (chemical linkage of peptides onto scaffolds) may be used to map conformational epitopes. The loose ends of the peptides are affixed onto synthetic scaffolds, so that the scaffolded peptide may be able to adopt the same spatial structure as the corresponding sequence in the intact protein. CLIPS technology is used to fix linear peptides into cyclic structures (‘single-loop’ format), and to bring together different parts of a protein binding site (‘double-loop’, ‘triple-loop’, etc. format), so as to create conformational epitopes that may be assayed for antibody binding (see, e.g., U.S. Pat. No. 7,972,993).

The epitopes bound by anti-GFRAL antibodies of the present disclosure may also be mapped using display techniques, including, for example, phage display, microbial display, and ribosome/mRNA display as described above. In these methods, libraries of peptide fragments are displayed on the surface of the phage or cell. Epitopes are then mapped by screening antibodies against these fragments using selective binding assays. A number of computational tools have been developed which allow the prediction of conformational epitopes based upon linear affinity-selected peptides obtained using phage display (see, e.g., Mayrose et al., Bioinformatics 23: 3244-3246, 2007). Methods are also available for the detection of conformational epitopes by phage display. Microbial display systems may also be used to express properly folded antigenic fragments on the cell surface for identification of conformational epitopes (see, e.g., Cochran et al., J. Immunol. Meth. 287: 147-158, 2004; Rockberg et al., Nature Methods 5: 1039-1045, 2008).

Methods involving proteolysis and mass spectroscopy may also be used to determine antibody epitopes (see, e.g., Baerga-Ortiz et al., Protein Sci. 2002 June; 11(6): 1300-1308). In limited proteolysis, the antigen is cleaved by different proteases, in the presence and in the absence of the antibody, and the fragments are identified by mass spectrometry. The epitope is the region of the antigen that becomes protected from proteolysis upon binding of the antibody (see, e.g., Suckau et al., Proc. Natl. Acad. Sci. USA 87:9848-9852, 1990). Additional proteolysis based methods include, for example, selective chemical modification (see, e.g., Fiedler et al., Bioconjugate Chemistry 1998, 9(2): 236-234, 1998), epitope excision (see, e.g., Van de Water et al., Clin. Immunol. Immunopathol. 1997, 85(3): 229-235, 1997), and the recently developed method of hydrogen-deuterium (H/D) exchange (see, e.g., Flanagan, N., Genetic Engineering and Biotechnology News 3(2): 25-28, 2010).

The epitope bound by anti-GFRAL antibodies of the present disclosure may also be determined by structural methods, such as X-ray crystal structure determination (see, e.g., WO 2005/044853), molecular modeling and nuclear magnetic resonance (NMR) spectroscopy, including NMR determination of the H-D exchange rates of labile amide hydrogens when free and when bound in a complex with an antibody of interest (see, e.g., Zinn-Justin et al. (1992) Biochemistry 31:11335-11347; Zinn-Justin et al. (1993) Biochemistry 32:6884-6891).

Additional antibodies binding to the same epitope as an anti-GFRAL antibody of the present disclosure may be obtained, for example, by screening of antibodies raised against GFRAL o for binding to the epitope, by immunization of an animal with a peptide comprising a fragment of human GFRAL comprising the epitope sequence, or by selection of antibodies using phage display for binding to the epitope sequence. Antibodies that bind to the same functional epitope might be expected to exhibit similar biological activities, such as blocking a biological activity of GFRAL, and such activities can be confirmed by functional assays of the antibodies.

4. Additional Activity Assays

In one embodiment, an anti-GFRAL antibody of the present disclosure is an antagonist antibody that inhibits a biological activity of GFRAL. The anti-GFRAL antibodies of the present disclosure may be assayed to determine if they inhibit a biological activity of GFRAL.

In one aspect, purified anti-GFRAL antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

In one embodiment, the present disclosure contemplates an altered antibody that possesses some but not all effector functions, which make it a desirable candidate for many applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In certain embodiments, the Fc activities of the antibody are measured to ensure that only the desired properties are maintained. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcγR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. An in vitro assay to assess ADCC activity of a molecule of interest is described, for example, in U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. To assess complement activation, a CDC assay, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be performed. FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art.

Although the foregoing present disclosure has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patent and scientific literatures cited herein are expressly incorporated in their entirety by reference.

EXAMPLES

The following are examples of methods and compositions of the present disclosure.

Example 1: Generation of Antibodies

Antibodies to a GFRALprotein were generated, for example, by immunizations of mice with cells expressing a GFRAL protein, co-expressing a RET protein and a GFRAL protein, or cross-linking of a GDF15 protein onto cells co-expressing a RETprotein and a GFRAL protein. Mice were also immunized with a GFRAL ECD, GDF15:GFRAL ECD complex and/or GFRAL ECD Fc fusion: RET ECD Fc fusion.

For example, the cells used for immunizations were prepared as follows. 293EXPI (Invitrogen) cells were transiently transfected with nucleic acid sequences encoding a GFRAL protein (SEQ ID NO: 1797) or co-transfected with nucleic acid sequences encoding a GFRAL protein and a RET protein (SEQ ID NOS: 1797 and 1813). Cells were analyzed for expression of GFRAL and RET by the respective specific antibodies by FACS. Cells were washed 2 times in PBS, pelleted by centrifugation and membrane preps were generated. 129/B6 or NZBW animals were immunized with membrane preps with adjuvants. Animals were boosted to induce a suitable titer. Titers were determined by ELISA and/or FACS. Single cell suspensions of lymphocytes were obtained from spleen and draining lymph nodes of animals with suitable titers. Cells were fused with SP2/0 myeloma cells at a ratio of 1:12 by electrofusion. Fused cells were plated into plates in the presence of HAT selection. After 10-14 days of culture, supernatants were collected and subjected to screening by cell imaging by CellnSight using GFRAL or GFRAL and RET overexpressing-293EXPI cells or by ELISA using GFRAL-Fc protein or RET and GFRAL Fc-heterodimers to confirm binding. Positive clones were further selected and subjected to subcloning.

In multiple campaigns of immunizations and fusions, over one-hundred thousand hybridoma clones were screened and more than two thousand clones were selected for GDF15-binding, cell-based GDF15-induced signaling and cell-based GDF15-independent signaling. Hundreds of clones (e.g., 250) were selected for additional study, including assays for binding affinity, domain mapping, and epitope specificity. Thousands of hybridoma supernatants were also tested in functional assays, including antagonistic and agonist activity assays. Hundreds of clones were purified for further testing.

Example 2: Screening and Selection of Antibodies

After 2 weeks of culture, hybridoma supernatants were screened for monoclonal antibodies binding to a GFRAL protein by cell imaging by CellnSight using GFRAL or GFRAL and RET overexpressing-293EXPI cells or by ELISA using GFRAL-Fc protein or RET and GFRAL Fc-heterodimers. Briefly, screening by cell imaging, 293EXPI cells were transiently transfected with nucleic acid sequences encoding GFRAL or co-transfected with nucleic acid sequences encoding GFRAL and RET. Transfected cells were plated onto 384-well plates with clear bottom. Media was replaced 24 hours post transfection. At 48 hrs, media was aspirated off the plates, hybridoma supernatants were added to the wells and incubated at room temperature for 30 mins. Then A647 anti-mouse Fc were added to the wells and incubated at room temperature for another 30 mins. Dapi positive cells were analyzed for a signal in the A647 channel and a positive A647 signal indicates GFRAL binders.

Briefly, screening by ELISA, GFRAL-Fc protein or RET and GFRAL Fc-heterodimers was captured by anti-human Fc reagents coated onto ELISA plates. Plates were blocked using PBS/1% BSA. 15 μL of hybridoma supernatants were added to the wells and incubated at room temperature for 1 hr. After 3 washes, 15 μL of HRP-anti-mouse Fc secondary were added to the wells and incubated at room temperature for 1 hr. After 3 washes, 15 μL of TMB were used to develop the plates. A positive signal indicates GFRAL binders.

From these assays, thousands of antibodies were identified as binders to GFRAL. These antibodies were subjected to further testing, which included assaying for binding affinity, domain mapping, epitope specificity, and agonistic and antagonistic function. Hundreds of antibodies were purified for further testing.

In addition, the binding affinity of antibodies to human and mouse GFRAL were measured. For example, antibodies were rank ordered based on their binding affinity to human GFRAL and mouse GFRAL by low resolution K_(D) measurement by Biacore. Briefly, an anti-mouse Fc antibody (Sigma-Aldrich, St. Louis, Mo.) was immobilized on all four flow cells of a CM5 chip using amine coupling reagents (GE Healthcare LifeSciences, Piscataway, N.J.). Purified antibodies were captured (˜100 RUs) on flow cells 2, 3 and 4 using flow cell 1 as a reference. This was followed by injection of human or mouse GFRAL (25 nM in PBS-P buffer) at a flow rate of 70 μL/min and monitoring the binding kinetics at 25° C.

Binding affinity measurements were also made in additional Biacore based assays. For example, equilibrium dissociation constant (K_(D)) measurements were carried out with purified antibodies to evaluate their binding to human GFRAL or mouse GFRAL. As mentioned above, anti-mouse Fc antibody (Sigma-Aldrich, St. Louis, Mo.) was immobilized on all four flow cells of a CM5 chip using amine coupling reagents (GE Healthcare LifeSciences, Piscataway, N.J.). Purified antibodies were captured (˜100 RUs) on flow cells 2, 3 and 4 using flow cell 1 as a reference. This was followed by injection of human or mouse GFRAL in PBS-P buffer) at a flow rate of 70 μL/min and the binding kinetics were evaluated at 25° C.

Representative results for binding affinity (e.g., K_(D) (nM)) to human and mouse GFRAL are shown in Table 26 below. In addition, representative results for off-rate of binding (e.g., K_(off) (1/s)) are shown in Table 26 below for exemplary antibodies.

TABLE 26 Binding Affinity Human GFRAL Mouse GFRAL Clone ID K_(D) (nM) k_(off) (1/s) K_(D) (nM) k_(off) (1/s) 1C1 <0.1  ~1E5 3.68  2.5E−3 25M22 <0.1  ~1E−5 4.2   1E−4 8D8 0.46 1.24E−4 8.17 2.81E−3 12A3 <0.1  ~1E−5 5.7 2.16E−3 3P10 <0.1  ~1E−5 <0.1  ~1E−5 5F12 <0.1  ~1E−5 2.2   9E−4 5A20 0.081 3.32E−5 <0.1  4−E4 17J16 <0.1  ~1E−5 <0.1   1E−5 6G9 <0.1  ~1E5 No N/A 2B8 0.73  ~1E5 3.76  1.3E−2 6N16 0.16 9.93E−5 ND NA 8C10 <0.1  7.2E−5 weak 49.26 2B11 0.1 8.45E−5 3.7 1.44E−3 1B3 <0.1  9.4E−9 4.4 1.07E−3 19K19 0.21 1.35E−4 4.7  3.4E−3 22N5 0.21 1.32E−4 3.47 9.43E−4 2A9 0.25 1.53E−4 3.6  2.9E−3 24G2 1.4 6.96E−4 5.02 1.73E−3 2I23 0.23 6.77E−5 ND NA 1A3 0.11 7.57E−5 ND NA P1B6 0.14 9.72E−5 ND NA P1H8 0.21 8.99E−5 ND NA P8G4 0.21 2.76E−4 ND NA NA = does not apply; ND = no binding detected

For exemplary antibodies 1C1 and 3P10, affinity is driven mainly by a very slow off-rate (see, e.g., FIG. 3). Antibodies that bind to GFRAL as shown in Table 26 above do not recognize (e.g., bind to) GNFR alpha 1, the most closely related homolog of GFRAL.

Example 3: Functional Assays

Antibodies to GFRAL generated, for example, such as described in Example 1, were tested for their functional activity in cell-based reporter assays.

For example, ELK1-luciferase reporter assays, which measure human GDF15 (hGDF15)-induced human GFRAL/RET signaling, were performed using transiently transfected HEK293T. The transfecting plasmids consisted of two reporter plasmids, Gal4-Elk1 and 5×UAS-Luc (Agilent Technologies PathDetect Elk1 trans-reporting system Cat #219005), and plasmids encoding human GFRAL (hGFRAL), cynomolgus monkey GFRAL (cynoGFRAL), mouse GFRAL (mGFRAL), or rat GFRAL (rGFRAL), and human RET (hRET), cynomolgus monkey RET (cynoRET), mouse RET (mRET) or rat RET (rRET). In these assays, hGDF15-induced activation of recombinantly expressed GFRAL/RET receptor complex in the cells triggers intracellular signaling transduction, which leads to ERK and then Elk1 phosphorylation. Once Gal4-Elk1 is phosphorylated, Gal4-Elk1 binds to the 5×UAS promoter region and turns on luciferase reporter gene transcription. The activity of luciferase is then measured in luciferase enzymatic assays.

Representative results for antibodies to GFRAL inhibiting human GFRAL/RET signaling are shown in Table 27 below.

TABLE 27 IC₅₀ (nM) Clone ID hGFRAL/hRET mGFRAL/mRET 5F12 0.834 6.276 3P10 4.088 1.029 17J16 0.6658 0.4075 6G9 2.65 ND 2B8 1.923 2.449 6N16 1.618 ND 8C10 1.786 WB 2B11 7.896 ND 25M22 2.887 1.113 12A3 4.993 0.9334 1B3 4.136 ND 19K19 3.877 0.8948 1C1 8.638 1.189 8D8 2.106 1.419 22N5 7.744 2.599 2A9 3.706 7.871 2B3 7.124 7.761 24G2 18.94 8.324 5A20 3.19 0.6 2I23 7.185 NP 1A3 ND NP P1B6 ND NP P1H8 ND NP P8G4 ND NP ND = blocking not detected in assay NP = assay not performed WB = weak blocking detected in assay

For some experiments, the above mentioned four plasmids (e.g., 2 reporter plasmids, GFRAL, RET) were transfected into newly harvested cells in suspension using FuGene6 transfection reagent (Promega). The GFRAL and RET DNA ratio in transfection was optimized for the each pair of receptors from indicated species and varied between 12:1 to 60:1. Transfected cells were seeded into 384-well plate (7500 cells/25 μL/well) in normal growth medium. After overnight incubation at 37° C., a mix of serially diluted antibodies and fixed concentration of hGDF15 were added. After 6 hrs at 37° C. incubation with the antibodies, an equal volume of Bright-Glo reagent (Promega) was added and luminescence signal was read using Enspire reader (Perkin Elmer).

Simultaneous addition of antibodies, antagonizing the hGDF15 effect, blocked hGDF15 signaling in a dose-dependent manner preventing expression of luciferase reporter gene.

Example 4: Additional Functional Assays

Anti-hGFRAL antibodies were tested for their hGDF15 antagonising activity in an additional cell-based assay, such as an U2OS assay stably expressing hGFRAL and hRET. One day before the assay, the cells were plated in 90 μl of DiscoveRx Assay Complete Cell Plating 16 Reagent (DiscoveRx, Cat #93-0563R16B) at 20K/each of 96 well plate. Next day the cells were treated with a mix of serially diluted antibodies and a fixed concentration of hGDF15 for 10 minutes at 37° C. Cis-bio Cellul'erk assay kit (Cat #64ERKPEH) was used to assay for ERK phosphorylation level following the manufacturer's protocol. Similar to the Hek293T Elk1 reporter assay, hGDF15 antagonising antibodies were able to prevent hGDF15-induced phosphorylation in a dose-dependent manner.

Representative results for antibodies to GFRAL preventing hGDF15-induced phosphorylation are shown in Table 28 below.

TABLE 28 Clone ID IC₅₀ (nM) 5F12 0.06 3P10 0.08 17J16 0.10 6G9 0.17 2B8 0.22 6N16 0.27 8C10 0.28 2B11 0.46 25M22 0.53 12A3 0.54 1B3 0.57 19K19 0.61 1C1 0.61 8D8 0.63 22N5 0.90 2A9 0.94 2B3 1.48 24G2 1.86 5A20 1.80 2I23 NP 1A3 NP P1B6 NP P1H8 NP P8G4 NP NP = assay not performed

Example 5: Ligand Competition, Domain Mapping and Epitope Binning

Antibodies that were selected for binding to GFRAL were evaluated in ligand competition binding assays, domain and epitope binning experiments.

Briefly, ligand competition binding assays were performed by capturing GFRAL-Fc protein or RET and GFRAL Fc-heterodimers onto ELISA plates by anti-human Fc. Plates were blocked using PBS/1% BSA. 15 μL of a dose titration of antibodies were added to the wells starting at 10 μg/mL with a 3× dilution and incubated at room temperature for 30 min. Without washing, biotinyated GDF15 (GFRAL ligand) was added to the wells for an additional 1 hr. After 3 washes, 15 μL of HRP-streptavidin secondary were added to the wells and incubated at room temperature for 1 hr. After 3 washes, 15 μL of TMB were used to develop the plate. A positive signal indicates that biotinylated GDF15 still binds to GFRAL captured on the plate and the antibody is not a ligand competitor. A negative signal indicates that biotinylated GDF15 no longer binds to GFRAL captured on the plate and the antibody is a ligand competitor.

Briefly, domain mapping assays were performed by transiently transfecting 293EXPI cells with nucleic acid sequences encoding GFRAL (SEQ, GFRAL domain 1, GFRAL domain 2, GFRAL domain 1+2, GFRAL domain 1+3, GFRAL domain 2+3, or GFRAL domain 3.

Seven GFRAL deletion constructs (Constructs 1 to 7) were tested, which included the following features contiguously from N-terminus to C-terminus: IgK signal sequence (depicted by lower case and underlined letters in the below sequences), FLAG tag sequence (depicted by lower case and italicized letters in the below sequences) and GFRAL protein sequences with various extracellular domain combinations (domain 1 depicted in bold capital letters; domain 2 depicted in underlined capital letters; domain 3 depicted in bold and underlined letters). Construct 1 (IgK-2Flag-GFRAL; SEQ ID NO: 1817) contained human GDF15 polypeptide (residues Q20 to L394), in which domains 1, 2 and 3 were present. Construct 2 (IgK-2Flag-GFRAL domain 1; SEQ ID NO: 1818) contained GFRAL-ΔD2,ΔD3 (residues Q20 to S130 and F317 to L394), in which domains 2 and 3 were deleted. Construct 3 (IgK-2Flag-GFRAL domain 2; SEQ ID NO: 1819) contained GFRAL-ΔD1,ΔD3 (residues S121 to C210 and F317 to L394), in which domains 1 and 3 are deleted. Construct 4 (IgK-2Flag-GFRAL domain 1+2; SEQ ID NO: 1820) contained GFRAL-ΔD3 (residues Q20 to C210 and F317 to L394), in which domain 3 was deleted. Construct 5 (IgK-2Flag-GFRAL domain 1+3; SEQ ID NO: 1821) contained GFRAL-ΔD2 (residues Q20 to S130 and C220 to L394), in which domain 2 was deleted. Construct 6 (IgK-2Flag-GFRAL domain 2+3; SEQ ID NO: 1822) contained GFRAL-ΔD1 (residues S121 to L394), in which domain 1 was deleted. Construct 7 (IgK-2Flag-GFRAL domain 3; SEQ ID NO: 1823) contained GFRAL-ΔD1,ΔD2 (residues A211 to L394), in which domains 2 and 3 were deleted.

Construct 1 (IgK-2Flag-GFRAL; SEQ ID NO: 1817) mdmrvpaqllqllllwlrqarc dykddddksaggdykddddkgg QTNNCTY LREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESN FQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGM WS CLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIP FNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPT CLSVIRSCQND ELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYID ILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSC FNYPTLSNVKGMALYT RKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKAR DPSSIQIPGEL Construct 2 (IgK-2Flag-GFRAL domain 1; SEQ ID NO: 1818) mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkgg QTNNCTY LREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESN FQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGM WSFNYPTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGI LLLVMVKLRTSRISSKARDPSSIQIPGEL Construct 3 (IgK-2Flag-GFRAL domain 2; SEQ ID NO: 1819) mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkggSHHGFKG MWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNI PFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCFNYPTLSNVKGMALYTRKH ANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPS SIQIPGEL Construct 4 (IgK-2Flag-GFRAL domain 1 + 2; SEQ ID NO: 1820) mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkgg QTNNCTY LREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESN FQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGM WS CLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIP FNIAQMLAFCDCAQSDIPCQQSKEALHSKTCFNYPTLSNVKGMALYTRKHA NKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKARDPSS IQIPGEL Construct 5 (IgK-2Flag-GFRAL domain 1 + 3; SEQ ID NO: 1821) mdmrvpaqllgllllwlrgarc dykddddksaggdvkddddkgg QTNNCTY LREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVESN FQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGM WS CLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLT CSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSC FNY PTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVM VKLRTSRISSKARDPSSIQIPGEL Construct 6 (IgK-2Flag-GFRAL domain 2 + 3; SEQ ID NO: 1822) mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkggSHHGFKG MWSCLEVAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNI PFNIAQMLAFCDCAQSDIPCQQSKEALHSKTCAVNMVPPPT CLSVIRSCQN DELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLTCSGSDDCKAAYI DILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSC FNYPTLSNVKGMALY TRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVMVKLRTSRISSKA RDPSSIQIPGEL Construct 7 (IgK-2Flag-GFRAL domain 3; SEQ ID NO: 1823) mdmrvpaqllgllllwlrgarc dykddddksaggdykddddkggAVNMVPP PT CLSVIRSCQNDELCRRHYRTFQSKCWQRVTRKCHEDENCISTLSKQDLT CSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHMLHRKSC FNY PTLSNVKGMALYTRKHANKITLTGFHSPFNGEVIYAAMCMTVTCGILLLVM VKLRTSRISSKARDPSSIQIPGEL

Cells were incubated with 1 μg/mL of antibodies for 30 mins at 4° C. After washing, cells were incubated with a fluorochrome labeled anti-mouse Fc secondary antibody either A488 or A647 for 30 mins at 4° C. After washing, cells were analyzed by FACs. A positive signal indicates that the antibody binds to the domain overexpressed by the transfected 293EXPI cells. A negative signal indicates that the antibody does not bind to the domain overexpressed by the transfected 293EXPI cells.

Briefly, epitope binning assays were performed by directly coating plates with 2 ug/mL of antibodies (mAb1). Plates were blocked using PBS/1% BSA. 2 μg/mL of antibodies (mAb2) were pre-incubated with 50 ng/mL of GFRAL-Fc protein for 30 min before adding to wells at room temperature for 1 hr. After 3 washes, 15 μL of HRP-anti-human Fc secondary were added to the wells and incubated at room temperature for 1 hr. After 3 washes, 15 μL of TMB were used to develop the plate. A positive signal indicates that GFRAL-Fc protein still binds to the captured antibody (mAb1) on the plate and antibody (mAb2) is not in the same epitope bin as the captured antibody (mAb1). A negative signal indicates that GFRAL-Fc protein no longer binds to the captured antibody (mAb1) on the plate and antibody (mAb2) is in the same epitope bin as the captured antibody (mAb1).

Representative results for inhibition of GDF15 binding to GFRAL, domain mapping of GFRAL and epitope binning are shown in Table 29 below.

TABLE 29 Clone ID Inhibition GFRAL Domain Epitope Bin 5F12 No D3 4 3P10 No D3 4 17J16 Partial D2 1 6G9 No D3 4 2B8 Yes — 2 6N16 No D3 4 8C10 Yes D2 2 2B11 No D3 4 25M22 Yes D2 2 12A3 Yes D2 2 1B3 No D3 4 19K19 Yes D2 2 1C1 Yes D2 3 8D8 Yes D2 1 22N5 Yes D2 3 2A9 Yes D2 3 2B3 Yes ND 1 24G2 Yes NA 1 5A20 Partial D2 1 2I23 No NA 4 1A3 No D1 5 P1B6 No D1 5 P1H8 No D1 7 P8G4 No D1 6 NA = does not apply; ND = no binding detected

As shown in Table 29 above, at least three classes of anti-GFRAL antibodies were identified, including antibodies that block GDF15 binding (e.g., competitive antagonists), antibodies that do not block GDF15 binding (e.g., non-competitive antagonists), and partial blockers of GDF15 binding. Antibodies were also identified as binding to domain 1, domain 2 or domain 3 of GFRAL. The antibodies that bound to domain 2 were found to either inhibit GDF15 binding to GFRAL or at leastly partically inhibit this binding. The antibodies that bound to domains 1 or 3 did not inhibit GDF15 binding to GFRAL.

An alignment of exemplary anti-GFRAL antibodies assayed in Examples 2-4 and described above is shown in FIGS. 4A-4B.

Alignments of VH and VL domains for antibodies that were found to bind domain 1, domain 2 or domain 3 are shown in FIGS. 5A-5F, respectively.

Example 6: Humanization

Humanized anti-GFRAL antibodies were generated, including from antibodies selected as described in Examples 1-5. Exemplary humanized antibodies are generated comprising one or more CDRs from Tables 1-24, including, for example, the CDRs for the antibody designated 1C1 (see, e.g., Table 1), for the antibody designated 25M22 (see, e.g., Table 8), for the antibody designated 17J16 (see, e.g., Table 7), for the antibody designated 5F12 (see, e.g., Table 4), and for the antibody designated 3P10 (see, e.g., Table 2). The sequences of VH and VL regions for exemplary humanized antibodies 1C1, 25M22, 17J16, 5F12, and 3P10 are shown in FIGS. 6A-6B, 7A-7B, 8A-8B, 9A-9B, and 10A-10B.

A number of anu-GFRAL antibodies were selected for sequencing and subsequently for humanization. An NCBI immunoglobulin (Ig) sequence blastp search was conducted to identify human germline sequences with significant similarity to mouse VH and VL sequences. Examples for potential human framework sequences are given in Table 30. Considering sequence similarities, biophysical properties and potential immunogenicity, germline sequences for IGH and IGK were chosen as human framework sequences. The germline sequences that were chosen are highlighted in bold in Table 30. In order to identify the best matching framework 4 sequence, a similar approach was taken for choosing JH and JK germline sequences, and these chosen sequences are also highlighted in bold in Table 30.

TABLE 30 Human Human Human Human germline germline germline germline donor donor donor donor sequence sequence sequence sequence Clone ID for VH for JH for VL for JK 3P10 IGHV1-18 IGHJ6 IGKV1-39 IGKJ4 IGHV1-3 IGHJ1 IGKV3-20 IGKJ2 IGHV1-46 IGKV2-30 IGHV1-2 IGK4-1 IGHV1-69 IGK3-11 5F12 IGHV1-46 IGHJ1 IGKV4-1 IGKJ4 IGHV1-3 IGHJ3 IGKV2-30 IGKJ2 IGHV1-69 IGKV1-39 IGHV1-2 IGKV3-11 IGKV1-5 IGKV1-27 25M22 IGHV5-51 IGHJ6 IGKV2-29 IGKJ4 IGHV1-46 IGHJ4 IGKV2-28 IGKJ2 IGHV1-3 IGKV2-18 IGHV1-2 IGKV2-30 IGHV1-69 IGKV3-20 IGHV1-18 IGKV1-39 1C1 IGHV4-39 IGHJ4 IGKV3-20 IGKJ4 IGHV4-31 IGHJ6 IGKV1-39 IGKJ2 IGHV3-33 IGKV2-30 IGHV4-59 IGKV2-28 17J16 IGHV1-2 IGHJ6 IGKV2-30 IGKJ4 IGHV1-46 IGKV2-29 IGKJ2 IGHV1-3 IGKV2-40 IGHV1-69 IGKV3-20 IGKV1-39 1A3 IGHV5-51 IGHJ6 IGKV2-28 IGKJ4 IGHV1-69 IGHJ4 IGKV2-30 IGKJ2 IGHV1-46 IGKV4-1 IGHV1-2 P1B6 IGHV5-51 IGHJ4 IGKV3-11 IGKJ4 IGHV1-69 IGHJ1 IGKV1-9 IGKJ2 IGHV1-46 IGKV1-5 IGHV1-2 IGKV3-15 IGKV1-33 IGKV3-20 P1H8 IGHV5-51 IGHJ6 IGHV2-28 IGKJ4 IGHV1-69 IGHJ4 IGHV2-30 IGKJ2 IGHV1-46 IGHV4-1 IGHV1-2 P8G4 IGHV4-59 IGHJ4 IGKV3-11 IGKJ1 IGHV3-23 IGHJ6 IGKV1-39 IGKJ2 IGHV4-4 IGKV1-5 IGHV3-30 IGKV3-20

The CDR sequences of murine antibodies were then transferred (e.g., grafted) to the corresponding positions of human IGH and IGK and J-region residues corresponding to framework four were added. The resulting protein sequence was back translated into a DNA sequence, codon optimized for expression in mammalian cells and synthesized (GeneArt/LifeTechnologies). Subsequently, the synthesized DNA fragment was cloned using In-Fusion technology (Clontech) into pTT5 vector (NRC Biotechnology Research Institute) to create expression ready constructs that contain a Kozak sequence, the hIgK signal peptide followed by the humanized variable-region of the desired antibody plus the constant region of the antibody (hIgG1/hIgK). At the same time individual residues in the framework regions were selected to be back-mutated to mouse residues in order to retain binding affinities. Special consideration was given to Vernier Zone residues. Such variants were either created by side directed mutatgenesis (QuickChange, Agilent) or de-novo DNA synthesis. Such expression constructs were then used for transient protein expression in Expi293 cells (Thermo Fischer), secreted antibodies were purified from tissue culture supernatant and tested for binding affinities. In order to optimize for binding affinity and lowest number of necessary back mutations, a second round of construct designs were typically made followed by antibody expression, purification and measurement of binding affinities.

Exemplary humanized anti-GFRAL antibodies include an antibody comprising: a VH that is SEQ ID NO: 1982 (HC-344e) and VL that is SEQ ID NO: 1997 (LC-344h), a VH that is SEQ ID NO: 1978 (HC-344a) and VL that is SEQ ID NO: 1992 (LC-344c); a VH that is SEQ ID NO: 1978 (HC-344a) and VL that is SEQ ID NO: 1997 (LC-344h); a VH that is SEQ ID NO: 1985 (HC-344h) and VL that is SEQ ID NO: 1997 (LC-344h); a VH that is SEQ ID NO: 1961 (HC-375d) and VL that is SEQ ID NO: 1976 (LC-375j); a VH that is SEQ ID NO: 1962 (HC-375e) and VL that is SEQ ID NO: 1976 (LC-375j); a VH that is SEQ ID NO: 1964 (HC-375g) and VL that is SEQ ID NO: 1967 (LC-375a); or a VH that is SEQ ID NO: 1964 (HC-375g) and VL that is SEQ ID NO: 1976 (LC-375j).

Example 7: Selection of Humanized Antibodies

Exemplary humanized anti-GFRAL antibodies identified in Example 6 were assayed for their binding affinity to human GFRAL. Binding affinity measurements were made in Biacore based assays. For example, equilibrium dissociation constant (K_(D)) measurements were carried out with purified antibodies to evaluate their binding to human GFRAL. Using methods described in Example II, anti-mouse Fc antibody (Sigma-Aldrich, St. Louis, Mo.) was immobilized on all four flow cells of a CM5 chip using amine coupling reagents (GE Healthcare LifeSciences, Piscataway, N.J.). Purified humanized anti-GFRAL antibodies were captured (˜100 RUs) on flow cells 2, 3 and 4 using flow cell 1 as a reference. This was followed by injection of human GFRAL in PBS-P buffer) at a flow rate of 70 μL/min and the binding kinetics were evaluated at 25° C.

Representative results for binding affinity (e.g., K_(D) (nM)) of exemplary humanized anti-GFRAL antibodies (e.g., humanized 3P10 and humanized 5F12) to human GFRAL are shown in Tables 31 and 32 below.

TABLE 31 Binding Affinity to Humanized anti-GFRAL Human GFRAL Antibodies K_(D) (pM) 3P10 Hybridoma <10 HC-344a/LC-344c 12 HC-344a/LC-344h <10 HC-344h/LC-344c <10 HC-344e/LC-344h <10 HC-344h/LC-344h <10

TABLE 32 Binding Affinity to Humanized anti-GFRAL Human GFRAL Antibodies K_(D) (pM) 5F12 Hybridoma 38 HC-375d/LC-375a 74 HC-375d/LC-375c 75 HC-375d/LC-375g 77 HC-375d/LC-375i 62 HC-375d/LC-375j 46 HC-375e/LC-375a 91 HC-375e/LC-375c 92 HC-375e/LC-375g 81 HC-375e/LC-375j 48 HC-375h/LC-375g 87 HC-375h/LC-375j 60 HC-375g/LC-375a 28 HC-375g/LC-375j 57

Example 8: Functional Assays of Humanized Antibodies

Exemplary humanized anti-GFRAL antibodies described in Examples 6 and 7 were tested for their functional activity in cell-based reporter assays similar to that described in Example 3.

For example, ELK1-luciferase reporter assays, which measure human GDF15 (hGDF15)-induced human GFRAL/RET signaling, were performed using transfected U20S and HEK293T. The transfecting plasmids consisted of two reporter plasmids, Gal4-Elk1 and 5×UAS-Luc (Agilent Technologies PathDetect Elk1 trans-reporting system Cat #219005), and plasmids encoding human GFRAL (hGFRAL and human RET (hRET). In these assays, hGDF15-induced activation of recombinantly expressed GFRAL/RET receptor complex in the cells triggers intracellular signaling transduction, which leads to ERK and then Elk1 phosphorylation. Once Gal4-Elk1 is phosphorylated, Gal4-Elk1 binds to the 5×UAS promoter region and turns on luciferase reporter gene transcription. The activity of luciferase is then measured in luciferase enzymatic assays.

Representative results for exemplary humanized antibodies to GFRAL (e.g., 3P10 and 5F12) inhibiting human GFRAL/RET signaling are shown in Table 33 and 34 below and in FIG. 11.

TABLE 33 Humanized anti-GFRAL Antibodies IC₅₀ (nM) 3P10 Hybridoma 4.02 HC-344a/LC-344c 7.26 HC-344a/LC-344h 4.63 HC-344h/LC-344c 29.4 HC-344e/LC-344h 6.95 HC-344h/LC-344h 5.7

TABLE 34 Humanized anti-GFRAL Antibodies IC₅₀ (nM) 5F12 Hybridoma 5.40 HC-375d/LC-375a 3.86 HC-375d/LC-375c 3.28 HC-375d/LC-375g 2.79 HC-375d/LC-375i 2.75 HC-375d/LC-375j 3.11 HC-375e/LC-375a 2.81 HC-375e/LC-375c 2.20 HC-375e/LC-375g 2.27 HC-375e/LC-375j 4.01 HC-375h/LC-375g 3.98 HC-375h/LC-375j 7.29

For some experiments, the above mentioned four plasmids (e.g., 2 reporter plasmids, GFRAL, RET) were transfected into newly harvested cells in suspension using FuGene6 transfection reagent (Promega). The GFRAL and RET DNA ratio in transfection was optimized for the each pair of receptors from indicated species and varied between 12:1 to 60:1. Transfected cells were seeded into 384-well plate (7500 cells/25 μL/well) in normal growth medium. After overnight incubation at 37° C., a mix of serially diluted antibodies and fixed concentration of hGDF15 were added. After 6 hrs at 37° C. incubation with the antibodies, an equal volume of Bright-Glo reagent (Promega) was added and luminescence signal was read using Enspire reader (Perkin Elmer).

Simultaneous addition of humanized anti-GFRAL antibodies, antagonizing the hGDF15 effect, blocked hGDF15 signaling in a dose-dependent manner preventing expression of luciferase reporter gene.

Example 9: Additional Functional Assays of Humanized Antibodies

Exemplary humanized anti-hGFRAL antibodies were tested for their hGDF15 antagonising activity in an additional cell-based assay, such as an U2OS assay stably expressing hGFRAL and hRET, as described in Example 4. One day before the assay, the cells were plated in 90 μl of DiscoveRx Assay Complete Cell Plating 16 Reagent (DiscoveRx, Cat #93-0563R16B) at 20K/each of 96 well plate. Next day the cells were treated with a mix of serially diluted antibodies and a fixed concentration of hGDF15 for 10 minutes at 37° C. Cis-bio Cellul'erk assay kit (Cat #64ERKPEH) was used to assay for ERK phosphorylation level following the manufacturer's protocol.

Similar to the Hek293T Elk1 reporter assay described in Example 4, humanized anti-hGFRAL antibodies (e.g., humanized 3P10) were able to prevent hGDF15-induced phosphorylation in a dose-dependent manner.

Representative results for humanized anti-GFRAL antibodies preventing hGDF15-induced phosphorylation are shown in Table 35 below and in FIG. 12.

TABLE 35 Humanized anti-GFRAL Antibodies IC₅₀ (nM) 3P10 Hybridoma 5.46 HC-344a/LC-344c 5.13 HC-344a/LC-344h 5.02 HC-344h/LC-344c 15.1 HC-344e/LC-344h 3.3 HC-344h/LC-344h 2.18

In a control experiment, GFRα1 (bound on chip surface) showed binding to its natural ligand GDNF (in solution, 50 nM) (see FIG. 13C).

Exemplary humanized anti-GFRAL antibodies (e.g. HC-344e+LC-344h) did not bind to receptor GFRα1, but showed high affinity binding to hGFRAL (FIGS. 13A and 13B) indicating specificity for GFRAL.

Example 10: Animal Studies

The effects of anti-GFRAL antibodies were evaluated in multiple animal studies.

A: Anti-GFRAL Antibodies Inhibit GDF15-Induced Weight Loss in DIO Mice (Acute)

To determine if an anti-GFRAL antibody is able to neutralize the GDF15-induced weight lowering effect in diet-induced obesity (DIO) mice, 17 week old male C57BL/6J DIO mice were used (Jackson Labs West, Sacramento, Calif.). Mice (41.3 g+0.4 g) were randomly assigned to receive an exemplary anti-GFRAL antibody (e.g., 1C1, 3P10, 17J16, 5A20, 25M22, 5F12, 8D8 and 12A3) or an anti-GDF15 antibody (e.g., 1M03). Each group had 6 mice per group. Each treatment group had its own PBS control group. The antibody dosage was 20 mg/kg. One day post injections with either PBS or antibody, the mice then received 3 consecutive daily injections of a GDF15 protein (e.g., having an amino acid sequence of SEQ ID NO:1811) at 0.5 mg/kg. Daily body weight and food intake were recorded (day 1-3 and day 7). Sartorius balance LE5201 was used for weighing. An automatic weighing record program (Sartorius YSW05 Software Wedge, Sartorius Mechatronics Corporation, 5 Orville Drive, Suite 200 Bohemia, N.Y. 11716) was used to transmit the weight data to a Microsoft Excel spreadsheet automatically.

Data are presented by mean±sem in term of raw body weight, delta body weight change and percentage of body weight change (i.e., % of delta body weight change over baseline body weight). A Student's t-test was used (two tails, two ends). In these experiments, P<0.05 was considered as statistically significant.

Exemplary anti-GFRAL antibodies, 1C1, 3P10, 17J16, 5A20, 25M22, 5F12, 8D8 and 12A3, are able to reverse GDF15-induced body weight loss and food intake reduction (FIGS. 14A and 14B). An anti-GDF15 antibody also reversed GDF15-induced body weight loss and food intake reduction in this model.

To determine if a non-competitive anti-GFRAL antibody or a competitive anti-GFRAL antibody block exogenous GDF15-induced weight lowering effect in a dose-dependent manner, 19 week old male C57BL/6J DIO mice were used. Initially mice (43.1 g±0.3 g) were randomly assigned to PBS (8 mice per group) or a GDF15 protein (e.g., having an amino acid sequence of SEQ ID NO: 1811) groups (0.1 mg/kg, 1.0 mg/kg, or 10 mg/kg, 24 mice per group). A GDF15 protein was confirmed to reduce body weight and food intake dose-dependently in the following day. Furthermore, the GDF15 treatment groups (0.1 mg/kg, 1.0 mg/kg, or 10 mg/kg) were randomly assigned to receive PBS, or an exemplary anti-GFRAL antibody (e.g., 1C1 or 3P10) (8 mice per group). Daily body weight and food intake were recorded (day 1-3 and day 7).

Exemplary anti-GFRAL antibodies reversed body weight reduction and food intake reduction that was induced by GDF15 (FIGS. 15 and 16).

B: Anti-GFRAL Antibodies Promote Body Weight Gain in DIO Mice (Chronic)

To determine if an exemplary anti-GFRAL antibody is able to increase body weight independent of exogenous GDF15 protein in a DIO model, 14 week old male C57BL/6J DIO mice were used (Jackson Labs West, Sacramento, Calif.). Mice (34.5 g±0.8 g) were randomly assigned to receive either PBS or exemplary anti-GFRAL antibodies (e.g., 1C1 or 3P10) at 10 mg/kg weekly. Each group had 10 mice per group. Daily body weight and food intake were recorded for 28 days. Sartorius balance LE5201 was used for weighing. An automatic weighing record program (Sartorius YSW05 Software Wedge, Sartorius Mechatronics Corporation, 5 Orville Drive, Suite 200 Bohemia, N.Y. 11716) was used to transmit the weight data to a Microsoft Excel spreadsheet automatically. Body composition was also evaluated by EchoMRI.

Exemplary anti-GFRAL antibodies increased body weight (by 6% and 7%, respectively) and food intake (by 3.4% and 3.7%, respectively) in DIO Mice (FIGS. 17 and 18). The increase in body weight by the exemplary anti-GFRAL antibodies is mainly attributed to increased fat mass (FIG. 19).

C: Anti-GFRAL Antibodies Reverse GDF-15-induced Loss of Body Mass in DIO Mice

To determine if an exemplary anti-GFRAL antibody is able to neutralize the GDF15-induced weight lowering effect in DIO mice, 17 week old male C57BL/6J DIO mice were used (Jackson Labs West, Sacramento, Calif.) and a recombinant adeno-associated virus (rAAV) expressing GDF15 was constructed.

Construction of the rAAV was performed as follows: Polymerase chain reactions (PCR) reagents kits with Phusion® high-fidelity DNA polymerase were purchased from New England BioLabs (F-530L, Ipswich, Mass.). The PCR reactions were set up according to manufacturer's instruction. Amplified DNA fragments containing an Igk signal peptide followed by a GDF15 encoding sequence was digested with restriction enzymes Spe I and Not I (the restriction sites were included in the 5′ or 3′ PCR primers, respectively) and were then ligated with AAV transgene vectors that had been digested with the same restriction enzymes. The vector used for expression contained a selectable marker and an expression cassette composed of a strong eukaryotic promoter 5′ of a site for insertion of the cloned coding sequence, followed by a 3′ untranslated region and bovine growth hormone polyadenylation tail.

The DIO mice were subjected to a high fat diet (Research Diets, catalog #D12492NI). The high fat diet contained 60 kcal % fat, 20 kcal % protein and 20 kcal % carbohydrate. The mice were administered a one-time tail vein injection of the rAAV described above or a control AAV vector expressing green fluorescent protein (GFP). 42 days post injection with rAAV expressing the GDF15 at 1×10¹⁰, mice were administered the exemplary anti-GFRAL antibody (3P10) or a control antibody (anti-KLH antibody) at a dose of 3 mg/kg. There were 20 mice per group. Mice body weight, lean and fat mass, energy expenditure and food intake were monitored over 12 weeks.

As shown in FIG. 20, the exemplary anti-GFRAL antibody (3P10) reversed the GDF15-induced loss of body weight and loss of fat and lean mass in DIO mice. Additionally, as shown in FIG. 21, the exemplary anti-GFRAL antibody reversed the GDF15-induced increase in energy expenditure and GDF15-induced reduction in food intake.

D: Anti-GFRAL Antibodies Reverse GDF-15-Induced Loss of Body Mass in Lean Mice (Chronic)

To determine if an exemplary anti-GFRAL antibody is able to neutralize the GDF15-induced weight lowering effect in lean mice, 15 week old male C57BL/6J mice were used (Jackson Labs West, Sacramento, Calif.). The mice were placed on a Chow Diet. As described in Example 11, part c, the mice were administered a one-time tail vein injection of the rAAV expressing GDF15 at 1×10¹⁰ or a control AAV vector expressing green fluorescent protein (GFP). 28 days post injection with rAAV expressing the GDF15, mice were administered the exemplary anti-GFRAL antibody (3P10) or a control antibody (anti-KLH antibody) at a dose of 3 mg/kg. There were 20 mice per group. Mice body weight, lean and fat mass, energy expenditure and food intake were monitored over 12 weeks.

As shown in FIG. 22, the exemplary anti-GFRAL antibody reversed the GDF15-induced loss of body weight, which included a reversal of both loss of fat mass and loss of lean mass in lean mice. Additionally, as shown in FIG. 23, the exemplary anti-GFRAL antibody reversed the GDF15-induced change in respitory exchange ratio (RER) and GDF15-induced reduction in food intake in lean mice.

E: Anti-GFRAL Antibodies do not Increase Body Weight Independent of GDF15-Induced Weight Loss in Lean Mice

To determine if an exemplary anti-GFRAL antibody is able to increase body weight independent of exogenous GDF15 protein in lean mice, 15 week old male C57BL/6J mice were used (Jackson Labs West, Sacramento, Calif.). Mice (26.1 g±0.3 g) were randomly assigned to receive either PBS or an exemplary anti-GFRAL antibody (e.g., 1C1 or 3P10) at 10 mg/kg weekly. Each group had 10 mice per group. Daily food intake and body weight were recorded in the first week, and then recorded weekly for another 3 weeks. Sartorius balance LE5201 was used for weighing.

Repeat doses of exemplary anti-GFRAL antibodies 1C1 and 3P10 do not significantly alter body weight and food intake in lean mice, although antibody 3P10 showed a weak trend to increase body weight (FIGS. 24 and 25).

F: Anti-GFRAL Antibodies Reverse Body Weight Loss and Increased Energy Expenditure in Mice with Chronic Kidney Damage

To determine if an exemplary anti-GFRAL antibody is able to reverse loss of body weight and increased energy expenditure in DIO mice with chronic kidney damage, 15 week old male C57BL/6J mice were used (Jackson Labs West, Sacramento, Calif.). After 5 weeks on HFD+adenine diet (0.3% @ induction phase for 10 days, then reducing to 0.1% @ maintenance phase for another 3 weeks), DIO Mice (26.1 g±0.3 g) were randomly assigned to receive either control antibody (anti-KLH) or exemplary anti-GFRAL antibodies (e.g., 3P10) at 1 mg/kg weekly. Each group had 12 mice per group. Daily body weight was recorded in the first week, and then recorded weekly for another 8 weeks. Sartorius balance LE5201 was used for weighing.

As shown in FIG. 26A, in order to verify the effect of adenine on kidney function, 10-day treatment with dietary adenine (0.25% adenine in HFD) was shown to induce kidney damage in mice. Dietary adenine also increased serum GDF15 levels (see FIG. 26B) and promoted weight loss (see FIG. 26C). Additionally, dietary adenine increased energy expenditure in mice (see FIG. 27A).

As shown in FIG. 27B, the exemplary anti-GFRAL antibody reversed the increased energy expenditure in mice with chronic kidney damage. Additionally, as shown in FIG. 28, the exemplary anti-GFRAL antibody reversed the loss of body weight and loss of fat and lean mass in mice with chronic kidney damage.

G: Humanized Anti-GFRAL Antibodies Inhibit GDF15-Induced Weight Loss in a Dose-Dependent Manner

To determine if an humanized anti-GFRAL antibody is able to neutralize the GDF15-induced weight lowering effect in diet-induced obesity (DIO) mice, 17 week old male C57BL/6J DIO mice were used (Jackson Labs West, Sacramento, Calif.). Initially mice (42 g±0.4 g) were randomly assigned to PBS (8 mice per group) or a GDF15 protein (56 mice per group). A GDF15 protein was confirmed to reduce body weight and food intake dose-dependently in the following day. Furthermore, 8 mice per group were randomly assigned to receive either an vehicle (anti-KLH) or an exemplary mouse anti-GFRAL antibody (e.g., m3P10) at a dosage of 1.0 mg/kg, or an exemplary humanized anti-GFRAL antibody (e.g., h3P10=HC-344e+LC-344h) at dosages of 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3 mg/kg or 10 mg/kg. Daily body weight was recorded (day 1-7). Sartorius balance LE5201 was used for weighing. An automatic weighing record program (Sartorius YSW05 Software Wedge, Sartorius Mechatronics Corporation, 5 Orville Drive, Suite 200 Bohemia, N.Y. 11716) was used to transmit the weight data to a Microsoft Excel spreadsheet automatically.

Data are presented by mean±sem in term of delta body weight change. A Student's t-test was used (two tails, two ends).

Exemplary humanized anti-GFRAL antibody HC-344e+LC-344h is able to inhibit GDF15-induced body weight loss in a dose-dependent manner (FIG. 29).

Example 11: Crystal Structure of GFRAL/GDF15 Complex

A: Complex Formation and Crystallization

A complex of a GFRAL protein and a GDF15 protein was made by mixing 1.2 molar excess of a GFRAL (W115-E351) protein with 1 molar GDF15 protein subunit (0.5 molar GDF15, which is a homodimer of two GDF15 subunits linked by a pair of disulfide bonds). The complex was purified by size exclusion chromatography to remove excess GFRAL. The GFRAL/GDF15 complex was crystallized by mixing 1 μL protein at 5 mg/ml with 0.5 μL reservoir solution and 0.5 μL seed in a crystallization drop, with the reservoir solution containing 1.0 mL of 0.1 M Bis-Tris pH 6.0, 1.5 M (NH₄)₂SO₄ and 10% ethylene glycol. The seed crystals were obtained from a crystallization condition including a reservoir solution of 0.1 M Bis-Tris pH 6.0 and 1.5 M (NH₄)₂SO₄. The crystallization setup was kept at room temperature in Rigaku 24 well clover leaf plate. The crystallization drop showed small needle crystals after three days of incubation.

An exemplary small needle crystal of a comples of a GFRAL protein and a GDF15 protein is shown in FIG. 30.

The molecular model was not available for GFRAL, hence NaBr soaking was used to determine crystal phasing. A GFRAL/GDF15 crystal obtained as described above was soaked with 0.5 M NaBr and 0.75 M NaBr containing reservoir solution. After 30 minutes, 0.5 M NaBr soaked crystals were in good condition, whereas 0.75 M NaBr soaking yielded cracked crystals. Crystals from both soaks and un-soaked crystals were mounted with 30% EG as a cryo-protectant.

The model described herein provides the first structural information for a GFRAL protein and the binding of a GFRAL protein to a GDF15 protein.

B: Data Collection and Structure Determination

GFRAL/GDF15 complex crystals were obtained and harvested from a 0.1 M Bis-Tris pH 6.0, 1.5 M (NH₄)₂SO₄ and 10% ethylene glycol reservoir condition as soaked and unsoaked crystals from 0.5 M and 0.7 M NaBr soaks. The crystals were treated with the mother liquor supplemented with 20% ethylene glycol as cryoprotectant and flash-frozen in liquid nitrogen. These crystals were then examined for x-ray diffraction at the synchrotron beamline IMCA-CAT, Advanced Photon Source, Argonne National Lab (ALS). The crystal diffracted up to 2.28-2.20 Å resolution.

X-ray diffraction statistics for exemplary GFRAL/GDF15 complex crystals are shown in Table 36.

TABLE 36 Data collection statistics Crystal I Wave length 0.9786 Å Space group P2₁ Unit cell (Å) a = 75.352 b = 88.768 c = 121.293 Resolution (Å)^(†) 50-2.20 (2.28-2.20) Number of measurements 118,710 Number of unique reflections  20,379 R_(sym) (%)^(†) 0.09 (0.58) Completeness (%)^(†) 97.4 (85.4) I/σ^(†) 18.9 (2.2) Redundancy^(†) 5.8 (4.9) Molecules in the A.U. 1 GFRAL 1 GDF15 ^(†)The parenthesis is for the highest resolution shell in Å.

Molecular replacement of GFRAL/GDF15 was performed by using the scaled dataset with a previously solved GFRAL/GDF15 complex at 3.2 Å resolutions as a starting model and the rigid body refinement (See Vagin, A. A., et al., (2004) “REFMAC5 dictionary: Organization of prior chemical knowledge and guidelines for its use.” Acta Crystallogr. D 60:2284-2295) and initial positional refinement was completed in REFMAC5 as implemented in CCP4. Several rounds of model rebuilding resulted in structures of the GFRAL/GDF15 complex.

Exemplary structures of a complex of a GFRAL protein and a GDF15 protein are shown, for example, in FIG. 31-39B.

Inspection of the initial electron density maps showed unambiguous density for GFRAL and GDF15. After rigid body refinement, several rounds of model building and restrained refinement were performed using COOT (See Emsley, P. and Cowtan, K. (2004) “COOT: model-building tools for molecular graphics.” Acta Crystallogr. D 60:2126-2132). After placement of the solvent molecules final refinement was completed.

The atomic coordinates from the x-ray diffraction patterns for the GFRAL/GDF15 complex are found in Table 49.

Refinement statistics for exemplary crystals are shown in Table 37.

TABLE 37 Refinement Statistics Refinement Range (Å) 35.82-2.20 R_(cryst) (%) 20.1 R_(free) (%) 26.2 Molecules GDF15, GFRAL; Water molecules 1, 1; 132 Bond lengths (Å) 0.019 Bond angles (°) 1.943 Average B-factors (Å²) Overall Main chain atoms (GDF15, GFRAL) 43.9, 57.6 Side chain atoms (GDF15, GFRAL) 51.2, 66.8 Water molecules 56.7 Ramachandran Plot (%) Overall Favored 96.3 Allowed 2.7 Disallowed 1.0

The clear electron density for GFRAL in an exemplary GFRAL/GDF15 complex crystal is illustrated in FIG. 31. FIG. 31 shows an electron density map (2fo−fc) for the GFRAL molecule calculated with 2.20 Å resolution data and contoured at the 1-level. The GFRAL residues are clearly visible.

C: Crystal Structure of GFRAL/GDF15 Complex

The crystal structure of a complex of a GFRAL protein and a GDF15 protein was determined.

Core interaction interface amino acids were determined as being the amino acid residues (on a protein such as GFRAL) with at least one atom less than or equal to 4.5 Å from the GFRAL interacting proteins (such as GDF15). 4.5 Å was chosen as the core region cutoff distance to allow for atoms within a van der Waals radius plus a possible water-mediated hydrogen bond.

Boundary interaction interface amino acids were determined as the amino acid residues (on a protein such as GFRAL) with at least one atom less than or equal to 5 Å from core interaction interface amino acids on GFRAL that interact with GFRAL interacting proteins (such as GDF15). Less than or equal to 5 Å was chosen as the boundary region cutoff distance because proteins binding to residues less than 5 Å away from core interaction interface amino acids on GFRAL will be within the van der Waals radius of GFRAL interacting proteins.

Amino acids that met these distance criteria were calculated with the Molecular Operating Environment (MOE) program from CCG (Chemical Computing Group).

FIG. 32 shows an exemplary illustration of a heterodimeric GFRAL/GDF15 complex, as found in the asymmetric unit of a GFRAL/GDF15 protein crystal. The dimeric molecule GDF15 has one intermolecular disulfide link, which was found to be weak due to radiation damage. One side of a GDF15 molecule can form a dimer in the asymmetric unit. FIG. 33 shows an exemplary dimeric arrangement of the GFRAL/GDF15 hetero dimers in a GFRAL/GDF15 crystal.

FIG. 34A-34B illustrate the extent of the protein-protein contacts on a GFRAL-GDF15 interface. The contact region on GFRAL is indicated by light gray arrows; the contact region on GDF15 is indicated by the black arrows.

FIG. 35 shows that three α-helices of GFRAL are involved in a GFRAL/GDF15 interface. Multiple disulfide bridges appear to stabilize the structural arrangement of the three GFRAL α-helices.

FIGS. 36A-36D illustrate different aspects of a GFRAL/GDF15 interface and the core and boundary amino acid residues of a GFRAL protein and a GDF15 protein involved in forming a GFRAL/GDF15 interface. The GFRAL protein and the GDF15 protein are depicted as ribbon diagrams with residues in the GFRAL/GDF15 interface shown in a space-filled surface representation. FIGS. 36A-36C show core interaction interface amino acids of the GFRAL protein and the GDF15 protein. FIG. 36D shows boundary interaction interface amino acids.

The amino acid sequence of a full-length precursor human GFRAL protein is shown below:

GFRAL sequences SEQ ID NO: 1797         10         20         30         40 MIVFIFLAMG LSLENEYTSQ TNNCTYLREQ CLRDANGCKH         50         60         70         80 AWRVMEDACN DSDPGDPCKM RNSSYCNLSI QVLVESNFQF          90        100        110        120 KECLCTDDFY CTVNKLLGKK CINKSDNVKE DKFKWNLTTR        130        140        150        160 SHHGFKGMWS CLEVAEACVG DVVCNAQLAS YLKACSANGN        170        180        190        200 PCDLKQCQAA IRFFYQNIPF NIAQMLAFCD CAQSDIPCQQ        210        220        230        240 SKEALKSKTC AVNMVPPPTC LSVIRSCQND ELCRRHYRTF        250        260        270        280 QSKCWQRVTR KCHEDENCIS TLSKQDLTCS GSDDCKAAYI        290        300        310        320 DILGTVLQVQ CTCRTITQSE ESLCKIFQHM LHRKSCFNYP        330        340        350        360 TLSNVKGMAL YTRKHANKIT LTGFHSPFNG EVIYAAMCMT        370        380        390 VTCGILLLVM VKLRTSRISS KARDPSSIQI PGEL 

GFRAL amino acids at the interface of the GFRAL/GDF15 complex are shown in Table 38.

TABLE 38 GFRAL Residues Binding GDF15* Core interaction Boundary interaction interface amino acids interface amino acids GLY140 SER156 LEU148 GLN147 ALA149 LEU148 ALA146 ALA149 VAL142 SER150 ASN145 TYR151 VAL139 LEU152 ALA135 LYS153 GLU136 ALA154 LEU152 CYS155 LEU132 PHE174 SER201 TYR175 ALA204 GLU136 LEU205 ALA137 LYS153 CYS138 ILE196 VAL139 PRO197 GLY140 GLN200 ASP141 VAL142 VAL143 CYS144 ASN145 ALA146 LEU186 CYS189 CYS191 ALA192 GLN193 SER194 ASP195 ILE196 PRO197 CYS198 GLN199 GLN200 SER201 LYS202 GLU203 ALA204 LEU205 HIS206 SER207 SER130 CYS131 LEU132 GLU133 VAL134 ALA135 *GFRAL amino acid numbering according to SEQ ID NO: 1797

The amino acid sequence of mature human GDF15 is shown below:

(SEQ ID NO: 1811) ARNGDHCPLG PGRCCRLHTV RASLEDLGWA DWVLSPREVQ VTMCIGACPS QFRAANMHAQ IKTSLHRLKP DTVPAPCCVP ASYNPMVLIQ KTDTGVSLQT YDDLLAKDCH CI

GDF15 residues at the interface of the GFRAL/GDF15 complex are shown in Table 39.

TABLE 39 Residues on GDF15 that bind to GFRAL* Core interaction Boundary interaction interface amino acids interface amino acids SER35 SER35 LEU34 VAL33 THR94 LEU34 GLY95 ASP93 GLN40 THR94 VAL96 GLY95 LEU98 VAL39 PRO36 GLN40 VAL87 ARG37 LEU88 GLU38 ILE89 VAL96 ASP102 SER97 THR100 LEU98 PRO85 PRO36 MET86 VAL87 LEU88 ILE89 VAL41 GLN90 LYS91 THR92 THR42 LEU104 LEU105 TYR101 ASP102 ASP103 GLN99 THR100 LEU24 TRP32 TRP29 ARG21 THR19 TYR83 ASN84 PRO85 MET86 VAL20 *GDF15 amino acid numbering according to SEQ ID NO: 1811

D: Model of GFRAL/RET/GDF15 Complex

The RET/GFRα1/GDNF ternary complex described by Goodman et al. (2014) CELL REPORTS 8, 1894-1904 (PDB 4UX8) was used as a template to build a model of the complex of GFRAL/GDF15/RET (from GFRAL/GDF15 structure, see, e.g., Examples 11-13). The RET/GFRα1/GDNF template resulted from an electron microscopy reconstruction of a reconstituted mammalian RET(ECD)-GDNF-GFRα1 ternary complex (Goodman et al., supra).

To compare the structural similarity of the GFRAL/GDF15 crystal structure from Example 12 and the structure of GFRα1/GDNF in the RET/GFRα1/GDNF template, the GFRAL structure in GFRAL/GDF15 crystal was superposed with GFRα1 in GFRα1/GDNF/RET model (PDB 4UX8) using MOE from CCG. The high quality of the superposition, and therefore the structural similarity of the GFRAL/GFRα1 and GFRAL/GDF15 complexes was demonstrated by an RMSD of GFRAL/GFRα1 backbone residues of 2.21 Å. This ternary complex model, including the GFRAL/GDF15 structure and the RET structure, was used to map the interactions between GFRAL and RET.

FIGS. 37A-37B illustrate exemplary aspects of the superposition of GFRAL and GFRα1 in 4XU8. RMSD of backbone residues was 2.21 Å.

FIGS. 38A-38D illustrate exemplary aspects of the interaction of a GFRAL protein with a RET protein in a RET/GFRAL/GDF15 model. In FIG. 38A, interacting GFRAL and GDF15 residues at the GFRAL/GDF15 interface as modeled are represented by stick models. In FIG. 38B, the RET-interacting residues on GFRAL are depicted in a space filled surface model. In FIG. 38C, the space filled surface model of the core interaction residues are highlighted on GFRAL and RET. In FIG. 38D, the space filled surface model of the boundary interaction residues are highlighted on GFRAL and RET.

FIGS. 39A-39B illustrate the core and boundary amino acid residues on a GFRAL protein identified in space filled surface models at the modeled RET interface. In FIG. 39A, core residues on GFRAL as modeled are shown in a darker grey in a space-filled surface model. In FIG. 39B, boundary residues on GFRAL as modeled are shown in a lighter grey in a space filled surface model.

Based on this modeling, a number of GFRAL residues were identified for interaction with RET residues, as shown in Table 40A. Additionally, a number of RET residues were identified for interaction with GFRAL residues, as shown in Table 40B.

TABLE 40A Residues on GFRAL that bind to RET in RET/GFRAL/GDF15 Model Core interaction Boundary interaction interface amino acids interface amino acids GLN246 ILE224 ARG247 ARG225 ARG250 GLN241 LYS251 SER242 CYS252 LYS243 ASP255 CYS244 GLU256 TRP245 ASN257 GLN246 CYS258 ARG247 ILE259 VAL248 SER260 THR249 THR261 ARG250 LEU262 LYS251 THR297 CYS252 GLN298 HIS253 SER299 GLU254 ASP255 GLU256 ASN257 CYS258 ILE259 SER260 THR261 LEU262 SER263 LYS264 GLN265 ASP266 LEU267 THR268 THR295 ILE296 THR297 GLN298 SER299 GLU300 GLU301 SER302 LEU303 ILE306 PHE307 MET310

TABLE 40B Residues on RET that bind to GFRAL in RET/GFRAL/GDF15 Model Core interaction Boundary interaction interface amino acids interface amino acids GLY74 ASP34 THR75 ALA35 TYR76 TYR36 ARG77 HIS71 THR78 TYR73 ASN113 LEU72 ARG114 GLY74 PHE116 TYR76 TYR122 THR75 GLN138 ARG77 ARG144 THR78 PRO305 ARG79 ALA306 LEU80 LEU310 LEU109 SER110 VAL111 ARG112 ASN113 GLY115 ARG114 PHE116 PRO117 LEU118 THR120 VAL121 TYR122 LEU123 LYS124 CYS137 GLN138 TRP139 PRO140 GLY141 CYS142 ALA143 ARG144 VAL145 TYR146 PHE147 ARG231 ASP264 ASP300 VAL303 VAL304 PRO305 ALA306 SER307 GLY308 GLU309 LEU310 ARG312 VAL311 ASN336

Example 12: Crystal Structures of GFRAL/Antibody Complexes

A1: Complex Formation and Crystallization of GFRAL/3P10/25M22 Fab Complex

A complex of a GFRAL protein, a 3P10 Fab and a 25M22 Fab (GFRAL/3P10/25M22 Fab complex) was formed by mixing GFRAL(W115-E351) with Fabs of 3P10 and 25M22 in a 1:1.2:1.2 molar ratio of GFRAL:3P10 Fab:25M22 Fab. The complex was purified from excess Fab molecules on a size exclusion chromatography column. The GFRAL/3P10/25M22 Fab complex was concentrated and crystallized as follows.

A volume of 1.5 mL of 3P10 Fab::GFRAL::25M22 Fab complex sample was concentrated by centrifugation to about 6.6 mg/mL using 10,000 MWCO Centricon® concentrator. The concentrated sample was immediately subjected to crystallization screening using 1 μL protein plus 1 μL reservoir per experiment. A first set of 96 conditions were set up that covered a factorial-based formulation sampling for the reservoir content. The crystallization setups were incubated at room temperature. Hampton Index and PegRx crystallization screens were used in the initial rounds of crystallization. The Hampton Index screen did not yield any potential hits, whereas the PegRx sreen yielded crystals under the following three crystallization conditions B11, D11, and H8:

-   -   B11: 0.1 M MES pH 6.0, 20% PEGMME 2000     -   D11: 0.1 M Imidazole pH 7.0, 12%, PEG 20,000     -   H8: 0.1 M TRIS pH 8.0, 16% PEG 10,000, 0.2 M Amonium Acetate.

Exemplary crystals of GFRAL/3P10/25M22 Fab complexes obtained under crystallization conditions B11, D11, and H8 are shown in FIG. 40.

To further optimize crystals for X-ray diffraction, GFRAL/3P10/25M22 complex was concentrated to 5.0 mg/mL and set up for crystallization and additional optimization, which led to improved crystals. Some improved crystals had a trapezium form. Some of the improved crystals were harvested, treated with a compatible cryoprotectant, and flash-frozen in liquid nitrogen. Identification and use of a compatible cryoprotectant was important to maintain sample crystallinity and to collect a high quality X-ray diffraction data set. Improved crystals obtained from a crystallization condition including 0.1 M Imidazole pH 7.0, 12% PEG 20,000 (D11) were treated with 35% ethylene glycol. Multiple crystals were flash-frozen in liquid nitrogen and X-ray diffraction data was collected at a synchrotron (ALS). One crystal diffracted to about 2.9 Å resolution. A full X-ray diffraction dataset was collected with this crystal.

The GFRAL/3P10/25M22 Fab complex crystal structure was determined. The position of the GFRAL component was clearly identified by molecular replacement based on the higher α-helix content in GRAL compared to 3P10 and 25M22 Fabs. In the exemplary crystal described here, one GFRAL and two Fab components (one 3P10 Fab and one 25M22 Fab) formed a ternary complex in the asymetric crystal unit.

Molecular replacement was used to identify Fabs in the GFRAL/3P10/25M22 Fab complex. PDB 1F8T, was the closest GFRAL homolog available for molecular replacement analysis (˜54% identity and ˜79% similarity to GFRAL). The structure solution clearly showed all Fab and GFRAL amino acid positions. The 1F8T GFRAL homolog structure was used as a stepping stone to solve the Fab and GFRAL components of the GFRAL/3P10/25M22 terniary complex crystal structure. In this structure, the stoichiometry in the was determined to be 1:1:1 3P10 Fab::GFRAL::25M22 Fab.

A2: Complex Formation and Crystallization of GFRAL/8D8/5f12 Fab Complex

A complex of a GFRAL protein, an 8D8 Fab and a 5F12 Fab (GFRAL/8D8/5F12 Fab complex) was formed by mixing GFRAL(W115-E351) with Fabs of 8D8 and 5F12 in a 1:1.2:1.2 molar ratio of GFRAL:8D8 Fab:5F12 Fab. The complex was purified from excess Fab molecules on a size exclusion chromatography column. The GFRAL/8D8/5F12 Fab complex was concentrated and crystallized as follows.

GFRAL/5F12/8D8 Fab complex was concentrated by centrifugation using 10,000 MWCO Centricon® concentrator. The concentrated sample was immediately subjected to crystallization screening using 1 μL protein plus 1 μL reservoir per experiment. A set of 96 conditions were initially set up that cover a factorial-based formulation sampling for the reservoir content. The crystallization setups were incubated at room temperature. Positive leads from initial screens were further optimized to yield high quality crystals that were amenable to diffraction analysis.

A volume of 1.5 mL of 5F12 Fab::GFRAL::8D8 Fab complex sample is concentrated by centrifugation to about 7.8 mg/mL using 10000 MWCO Centricon concentrator. Immediately upon completion, this sample is subjected to crystallization screening using 1 μL protein plus 1 μL reservoir per experiment. A set of 96 conditions are initially setup that cover a factorial-based formulation sampling for the reservoir content. These setups are incubated at room temperature. Initially, Hampton Index and PegRx crystallization screens were used for crystallization, Index and PegRx gave crystals in the following three crystallization conditions C6, E11, and C2:

-   -   C6: 0.1 M Bis-Tris pH 6.5, 14% PEG 3350     -   E11: 1.7 M AmSO4, 0.1 M Bis-Tris pH 6.5, 3% PEG MME 550     -   C2: 0.1 M Imidazole pH 7.0, 20% Jeffamine 2001.

Preliminary crystals were obtained as shown in FIG. 41.

Optimization of PegRx C6 and C2 crystallizing conditions was conducted by micro seeding technique, which lead to suitable diffraction mono-crystals. These suitable diffraction mono-crystals have a hexagonal bipyramid form obtained in 10% Ethylene glycol with 0.1 M Imidazole pH 7.0, 20% Jeffamine 2001 pH 7.0. Some of these crystals were harvested; treated with a compatible cryoprotectant; and flash-frozen in liquid nitrogen. Use of a compatible cryoprotectant is important to maintain the sample crystallinity, which results in a workable data set. The crystals were treated 20% MPD obtained from 0.1 M Imidazole pH 7.0, 20% Jeffamine 2001 pH 7.0.

B1: Data Collection and Structure Determination of GFRAL/3P10/25M22 Fab Complex

X-Ray diffraction data was collected for twenty crystals of the GFRAL/3P10/25M22 Fab complex at a synchrotron (ALS). The first ten crystals yielded one complete data set with a resolution of 3.17 Å. Ten additional crystals that were further optimized diffracted up 2.9 Å, and this data set was used for structure determination and refinement. The X-ray diffraction data was indexed using DENZO and subsequently integrated and scaled with SCALEPACK from the program suite HKL2000. See Otwinowski Z. and Minor W. “Processing of X-ray Diffraction Data Collected in Oscillation Mode,” METHODS IN ENZYMOLOGY, Volume 276: Macromolecular Crystallography, part A, p. 307-326, 1997, C. W. Carter, Jr. & R. M. Sweet, Eds., Academic Press (New York). The X-ray diffraction of the selected crystal was identified as having an orthorhombic Bravais lattice symmetry. The space group was determined to be P2₁2₁2₁ based on the systematic absences along (h, 0, 0), (0, k, 0), and (0, 0, l) axes. Analysis of the Matthew's coefficient suggests the crystal's asymmetric unit may accommodate one assembly with approximately 124.4 kDa and a corresponding solvent content of 56%.

X-ray diffraction statistics for exemplary GFRAL/3P10/25M22 Fab complex crystals are shown in Table 41.

TABLE 41 Data collection statistics Crystal I Wavelength 0.9774 Å Space group P2₁2₁2₁ Unit cell (Å) a = 52.500 b = 116.045 c = 227.048 Resolution (Å)^(†) 50-2.91 (3.01-2.91) Number of measurements 227,808 Number of unique reflections  31,548 R_(sym) (%)^(†) 0.11 (0.86) Completeness (%)^(†) 100 (100) I/σ^(†) 17.8 (2.7) Redundancy^(†) 7.2 (7.2) Molecules in the A.U. 2 Fabs 1 GFRAL ^(†)The parenthesis is for the highest resolution shell in Å.

Molecular replacement of the GFRAL/3P10/25M22 Fab Complex was performed by using the scaled dataset with the previously solved GFRAL (see Example 12, part C) and the 79% homologous GFRAL structure of 1F8T as starting models within the program PHASER. Data extending from 40-3.5 Å resolution gave one GFRAL and one Fab. Molrep yielded one more Fab in the structure solution. The solution had a complex of one GFRAL and two Fabs in the asymmetric unit. The solution was refined using REFMAC. COOT was used for model building. The refinement density clearly indicates missing loops in Fabs and GFRAL. After placement of a few solvent molecules final refinement was completed.

The atomic coordinates from the x-ray diffraction patterns for the GFRAL/3P10/25M22 Fab Complex are found in Table 50.

Exemplary refinement statistics of a GFRAL/3P10/25M22 Fab Complex crystal structure are shown in Table 42.

TABLE 42 Refinement Statistics Refinement Range (Å) 47.7-2.9 R_(cryst) (%) 23.6 R_(tree) (%) 31.0 Molecules 3P10 Fab::GFRAL::25M22 Fab 1, 1, 1 Bond lengths (Å) 0.011 Bond angles (°) 1.651 Average B-factors (Å²) Overall Main chain atoms ( GFRAL) 66.3 Side chain atoms (GFRAL) 71.6 Main chain atoms (3P10) Hc:Lc 92.7; 75.5 Side chain atoms (3P10) Hc:Lc 89.8; 78.5 Main chain atoms (25M22) Hc:Lc 85.5; 101.9 Side chain atoms (25M22) Hc:Lc 83.4; 101.2 Water molecules 67.1 Ramachandran Plot (%) Overall Favored 78.3 Allowed 20.6 Disallowed 1.2

The clear electron densities of CDR regions in each chain of the 3P10 and 25M22 Fab fragments in the GFRAL/3P10/25M22 Fab complex are illustrated in FIG. 42 (electron density 2mFo-DFc weighted, 1.06 contour level). The 3P10 and 25M22 chains were identified clearly in the electron density, with the exception of region 131-137 in the 3P10 heavy chain (Chain-H), where a few terminal residues were not identified in the electron density.

B2: Data Collection and Structure Determination of GFRAL/8D8/5F12 Fab Complex

The crystals were examined at the synchrotron at APS. Twenty five such crystals were examined for x-ray diffraction. Sixteen crystals were send first time to synchrotron which yielded a diffraction >5 Å and the second round of optimized crystals were diffracted up 3.5 Å, which is used for structure determination and refinement. The X-ray diffraction data were indexed using DENZO and integrated and scaled with SCALEPACK from the program suite HKL2000 (Otwinowski and Minor, 1997). The X-ray diffraction of the selected crystal was identified as having an orthorhombic Bravais lattice symmetry. The space group was determined to be P6522 based on the systematic absences along (h, 0, 0) axes. Analysis of the Matthew's coefficient suggests the crystal's asymmetric unit may accommodate one assembly with approximately 246.6 kDa and a corresponding solvent content of 61%. The structure solution was checked with enantiomeric spacegroup P6122, which gave very poor Rfactor and Rfree during the rigid body and restraint refinement.

X-ray diffraction statistics for the GFRAL/8D8/5F12 complex crystals are shown in Table 43.

TABLE 43 Data collection statistics Crystal I Wavelength 0.9787 Å Space group P6₅22 Unit cell (Å, °) a = 133.021 b = 133.021 c = 564.819 γ = 120 Resolution (Å)^(†) 50-3.55 (3.68-3.55) Number of measurements 524,288 Number of unique reflections  37,081 R_(sym) (%)^(†) 0.13 (1.00) Completeness (%)^(†) 100 (100) I/σ^(†) 22.0 (3.4) Redundancy^(†) 14.1 (14.6) Molecules in the A.U. 4 Fabs 2 Receptor ^(†)The parenthesis is for the highest resolution shell in Å.

Molecular replacement of Fabs/Receptor was performed by using the scaled dataset with the previously solved of fab pdb:31U4 used for 8D8 (89% homologous) and pdb:4M7K used for 5F12 (84% homologous) were used as a starting models within the program PHASER using the data extending from 40-5.0 Å resolution gave two assembly of one receptor and two Fabs. The solution had two receptors and four Fabs complex in the asymmetric unit. The solution was refined used REFMAC and Coot was used for model building.

The structure solution was checked with enantiomeric spacegroup P6₁22. The Molrep gave two receptors and four Fabs complex. The Molrep model further submitted to Refmac for refinement end up with very poor R_(factor) and R_(free) during the rigid body and restraint refinement which confirms that the current space group is P6₅22.

The atomic coordinates from the x-ray diffraction patterns for the GFRAL/8D8/5F12 Fab Complex is found in Table 51.

Exemplary refinement statistics of a GFRAL/8D8/5F12 Fab Complex crystal structure are shown in Table 44.

TABLE 44 Refinement Statistics Refinement Range (Å) 49.2-3.55 R_(cryst) (%) 25.1 R_(free) (%) 35.8 Molecules 3P10 Fab::GFRAL::25M22 2, 2, 2 Bond lengths (Å) 0.011 Bond angles (°) 1.63

The clear electron density of one GFRAL protein with the 8D8 and 5F12 Fab fragments (two Fab chains) in the GFRAL/8D8/5F12 Fab complex is illustrated in FIG. 43 (electron density 2mFo-DFc weighted, 1.06 contour level). The refinement density clearly indicates most of the loops in Fabs and receptor molecule. The 5F12 and 8D8 chains were identified clearly in the electron density except for the region between amino acids 139-142 in chain-I (5F12 vH), 132-136 in chain-R (8D8 vH) and 131-136 in chain-U (8D8 vL), as well as a few terminal residues were not identified in the density.

C1: Crystal Structure of GFRAL/3P10/25M22 Fab Complex

The crystal structure of a 3P10 Fab::GFRAL::25M22 Fab complex was determined.

3P10 Fab and 25M22 Fab amino acid sequences are shown below, with VH and VL sequences bolded and CDR regions bolded and underlined.

3P10 Fab Hc: (SEQ ID NO: 1824) QIQLVQSGPELKKPGETVKISCKAS GYTFTDYGVI WVKQAPGKALKWMG WI NTYTGEPTYADDLKG RFAFSLETSASSASLQINNLKNEDTATYFCAR RYGP EDIDY WGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDEVD 3P10 Fab Lc: (SEQ ID NO: 1825) DIVLTQSPVSLAVSLGQRATISC RASESVDNYGISFMS WFQQKPGQPPKLL IY AASHQGS GVPARFSGSGSGTDFSLNIHPMEEDDSAMYFC LQSKEVPWT F GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC 25M22 Fab Hc: (SEQ ID NO: 1826) QVQLQQSGPDLVKPGASVKISCKAS GYTFTSYWVN WMKQRPGKGLEWIG RI YPGDGDTNYNGKFKG KATLTADKSSSTAYMQLSSLTSEDSAVYFCAR AYLL RLRRTGYYAMDY WGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPK SCDEVD 25M22 Fab Lc: (SEQ ID NO: 1827) DVVLTQTPLSLPVNIGDQASISC KSTKSLLNSDEFTYLD WYLQKPGQSPQL LIF LVSNRFS GVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQSNYLPY T FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC

FIG. 44 illustrates an aspect of a 3P10 Fab::GFRAL::25M22 Fab complex by a ribbon diagram. The Fab fragments interact with an asymmetric unit of GFRAL in the 3P10 Fab::GFRAL::25M22 Fab complex crystal. The crystal structure also showed that GFRAL epitope residues 290-312 were presented to the 3P10 antibody and GFRAL N-terminal epitope residues 130-157 were presented to the 25M22 antibody heavy chain CDR regions.

FIG. 45 illustrates CDR regions of 3P10 and 25M22 Fabs with interacting GFRAL and Fab residues highlighted in boxes.

FIGS. 46-48 illustrate aspects of GFRAL interactions with 3P10 and 25M22L Fabs in ribbon diagrams with select amino acid residues shown as stick models. For example, FIG. 46 illustrates aspects of a GFRAL epitope in the vicinity of the 3P10 heavy chain CDR region. The CDR sequences of 3P10 Hc are shown in FIG. 45. As another example, FIG. 47 illustrates aspects of a GFRAL epitope in the vicinity of the 3P10 light chain CDR region. The CDR sequences of 3P10 Lc are shown in FIG. 45. As another example, FIG. 48 illustrates aspects of a GFRAL epitope in the vicinity of the 25M22 heavy chain CDR region. The CDR sequences of 25M22 Lc are shown in FIG. 45.

An analysis of the GFRAL epitope presented to 25M22 Fab in the 3P10 Fab::GFRAL::25M22 Fab complex crystal structure showed that 25M22's mechanism of action is that of a competitive GDF15 inhibitor and involves blocking GDF15 binding to GFRAL. Exemplary core interaction interface amino acids on the GFRAL protein and on the 25M22 Fab CDRs for the heavy and light chains are shown in FIG. 49.

FIGS. 50A and 50B illustrates the core amino acids residues in the GFRAL/25M22 Fab interaction interface. FIG. 50A illustrates the structure of GFRAL with the core 25M22 interaction interface amino acids (binding epitope) on GFRAL highlighted in a space-filled surface model. Core interface amino acids on 25M22 CDRs for GFRAL are also highlighted in a space-filled surface model. FIG. 50B illustrates the structure of GFRAL in a ribbon diagram with core 25M22 Fab interaction interface amino acids on GFRAL (GFRAL epitope residues) highlighted in a space-filled surface model.

FIG. 51 illustrates boundary interaction interface amino acids on GFRAL for GFRAL/25M22 Fab binding. Boundary of interaction interface amino acids on GFRAL (for 25M22 Fab binding) are highlighted as space-filled surface models.

FIGS. 52-53 illustrate the overlap of GFRAL epitopes for 25M22 Fab and GDF15 binding on GFRAL. Two side views of a GFRAL ribbon diagram are shown in the left and right panels of FIG. 52. FIG. 53 shows a top view of the overlapping GFRAL epitopes for 25M22 Fab and GDF15 binding. Core interaction interface amino acids residues on GFRAL for 25M22 Fab and GDF15 binding are highlighted in a space-filled surface model. Light gray surface shading represents GFRAL surface covered by 25M22 Fab, whereas dark gray surface shading (highlighted by black arrows) shows GFRAL surface covered by GDF15.

GFRAL amino acids at the interface of the GFRAL/25M22 Fab complex are shown in Table 45. To compare the interaction interface amino acids in GFRAL/25M22 Fab and GFRAL/GDF15 complexes, Table 45 further lists the GFRAL amino acids at the interface of the GFRAL/GDF15 complex, which is also shown in Table 38. Table 45 illustrates that all core interaction interface amino acid residues on GFRAL that bind to GDF15 are also core interaction interface amino acids in the GFRAL/25M22 Fab interaction.

The amino acid sequence of a full-length precursor human GFRAL protein is shown below (see also Example 12, part C):

GFRAL sequences SEQ ID NO: 1797         10         20         30         40 MIVFIFLAMG LSLENEYTSQ TNNCTYLREQ CLRDANGCKH         50         60         70         80 AWRVMEDACN DSDPGDPCKM RNSSYCNLSI QVLVESNFQF          90        100        110        120 KECLCTDDFY CTVNKLLGKK CINKSDNVKE DKFKWNLTTR        130        140        150        160 SHHGFKGMWS CLEVAEACVG DVVCNAQLAS YLKACSANGN        170        180        190        200 PCDLKQCQAA IRFFYQNIPF NIAQMLAFCD CAQSDIPCQQ        210        220        230        240 SKEALHSKTC AVNMVPPPTC LSVIRSCQND ELCRRHYRTF        250        260        270        280 QSKCWQRVTR KCHEDENCIS TLSKQDLTCS GSDDCKAAYI        290        300        310        320 DILGTVLQVQ CTCRTITQSE ESLCKIFQHM LHRKSCFNYP        330        340        350        360 TLSNVKGMAL YTRKHANKIT LTGFHSPFNG EVIYAAMCMT        370        380        390 VTCGILLLVM VKLRTSRISS KARDPSSIQI PGEL

TABLE 45 Residues on GFRAL that bind to GDF15 Residues on GFRAL that bind to 25M22 Core interaction Core interaction Boundary interaction interface amino acids interface amino acids interface amino acids LEU132 LEU132 SER130 ALA135 ALA135 CYS131 GLU136 GLU136 LEU132 VAL139 VAL139 GLU133 GLY140 GLY140 VAL134 VAL142 VAL142 ALA135 ASN145 ASN145 GLU136 ALA146 ALA146 ALA137 LEU148 LEU148 CYS138 ALA149 ALA149 VAL139 LEU152 LEU152 GLY140 LYS153 LYS153 ASP141 ILE196 ILE196 VAL142 PRO197 PRO197 VAL143 GLN200 GLN200 CYS144 SER201 SER201 ASN145 ALA204 ALA204 ALA146 LEU205 LEU205 GLN147 LEU148 ALA149 SER150 TYR151 LEU152 LYS153 ALA154 CYS155 SER156 PHE174 TYR175 LEU186 CYS189 CYS191 ALA192 GLN193 SER194 ASP195 ILE196 PRO197 CYS198 GLN199 GLN200 SER201 LYS202 GLU203 ALA204 LEU205 HIS206 SER207 *GFRAL amino acid numbering according to SEQ ID NO: 1797

An analysis of the GFRAL epitope presented to 3P10 Fab in the 3P10 Fab::GFRAL::25M22 Fab complex crystal structure showed that 3P10's mechanism of action is that of a non-competitive GDF15 inhibitor and does not involve blocking GDF15-binding to GFRAL. Exemplary core interaction interface amino acids on the GFRAL protein and on the 3P10 Fab CDRs for the heavy and light chains are shown in FIG. 54.

FIGS. 55A-55B illustrate the interaction interface residues on GFRAL and 3P10 Fab as stick models (FIG. 55A) or a space-filled surface models (FIG. 55B).

FIGS. 56A-56B illustrates core (FIG. 56A) and boundary (FIG. 56B) interaction interface amino acids on GFRAL for 3P10 Fab binding (GFRAL structural 3P10 binding epitope) in a space-filled surface model.

FIG. 57 illustrates the partial overlap of GFRAL epitopes for 3P10 Fab and RET on GFRAL. Two side views of a GFRAL ribbon diagram are shown in the left and right panels of FIG. 57. Interaction interface amino acids residues on GFRAL for 3P10 Fab and RET binding are highlighted in a space-filled surface model.

FIG. 58 illustrates the overlap of GFRAL epitopes for 25M22 Fab and 25M22 Fab binding on GFRAL. Top and a bottom views of a GFRAL ribbon diagram are shown on the left (top view) and right (bottom view) panels of FIG. 58. Boundary interaction interface residues on GFRAL involved in 25M22 Fab and 25M22 Fab binding are shown as space-filled surface models.

GFRAL amino acids at the interface of the GFRAL/3P10 Fab complex are shown in Table 46. To compare the interaction interface amino acids in GFRAL/3P10 Fab and GFRAL/RET complexes, Table 446 further lists the GFRAL amino acids at the interface of the GFRAL/RET complex, which is also shown in Table 40A. Table 46 shows core interaction interface amino acid residues on GFRAL that bind to both RET and 3P10 Fab in bold.

TABLE 46 Residues on GFRAL that bind to RET in RET/GFRAL/GDF15 Residues on GFRAL that bind to Model 3P10 in 3P10/GFRAL structure Core interaction Core interaction Boundary interaction interface amino acids interface amino acids interface amino acids Gln246 MET214 LEU164 Arg247 PRO216 LYS208 Arg250 PRO217 VAL212 Lys251 GLN290 ASN213 Cys252 CYS291 MET214 Asp255 THR292 VAL215 Glu256 CYS293 PRO216 Asn257 ARG294 PRO217 Cys258 THR295 PRO218 Ile259 ILE296 THR219 Ser260 THR297 CYS220 Thr261 GLN298 LEU221 Leu262 SER299 VAL223 Thr297 GLU301 TRP245 Gln298 LYS305 LEU267 Ser299 GLN308 CYS269 HIS309 GLN288 HIS312 VAL289 SER315 GLN290 CYS291 THR292 CYS293 ARG294 THR295 ILE296 THR297 GLN298 SER299 GLU300 GLU301 SER302 LEU303 CYS304 LYS305 ILE306 PHE307 GLN308 HIS309 MET310 LEU311 HIS312 ARG313 LYS314 SER315 CYS316 PHE317 Core interaction interface amino acid residues on GFRAL that bind to both RET and 3P10 Fab are shown in bold.

C1: Crystal Structure of GFRAL/8D8/5F12 Fab Complex

The crystal structure of a 8D8 Fab::GFRAL::5F12 Fab complex was determined.

8D8 Fab Hc: (SEQ ID NO: 1828) QVQLKESGPGLVAPSQSLSITCTVS GFSLSRYSVH WVRQPPGKGLEWLG MI WGFGSTDYNSALKS RLSITKDNSKSQFFLKMNSLQTDDTAMYYCAR IHTTA GSY WGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDEVDG 8D8 Fab Lc: (SEQ ID NO: 1829) DIVMTQSQKFMSTSIGDRVSVTC KASQNVGTNVA WYQQKPGQSPKALVY ST SYRYS GVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC HQYNSYPLT FGAGT KLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 5F12 Fab Hc: (SEQ ID NO: 1830) QVQLKQSGTELVRPGASVKLSCKAS GYTFTDYYIN WVKQRPGQGLEWIA RI YPGNGNTYHNEKFKG KATLTAEKSSSTAYMQLSSLTSEDSAVYFCAR EGLY YDYDRYFDY WGQGTALTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI CNVNHKPSNTKVDKKVEPKSCDEVDG 5F12 Fab Lc: (SEQ ID NO: 1831) NIVLTQSPASLAVSLGQRATISC RASESVDTYGNSFMH WYQQKPGQPPKLL IY LASNLES GVPARFSGSGSRTDFTLTIDPVEADDAATYYC HQNNEDPPA F GGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

FIG. 59 illustrates an aspect of a 3P10 Fab::GFRAL::25M22 Fab complex by a ribbon diagram. The Fab fragments interact with an asymmetric unit of GFRAL in the 3P10 Fab::GFRAL::25M22 Fab complex crystal. The crystal structure also showed that GFRAL epitope residues 290-312 were presented to the 3P10 antibody and GFRAL N-terminal epitope residues 130-157 were presented to the 25M22 antibody heavy chain CDR regions

FIG. 60 illustrates 8D8 Fab and 5F12 Fab binding sites on the GFRAL protein. Residues on the GFRAL protein that are important for Fab bindings are shown as stick models.

An analysis of the GFRAL epitope presented to 8D8 Fab in the 8D8 Fab::GFRAL::5F12 Fab complex crystal structure showed that 8D8's mechanism of action is that of a competitive GDF15 inhibitor and involves blocking GDF15 binding to GFRAL. Exemplary core interaction interface amino acids on the GFRAL protein and on the 8D8 Fab CDRs for the heavy and light chains are shown in FIG. 61.

FIGS. 62A, 62B, 62C and 62D illustrate the core and boundary amino acid residues in the GFRAL/8D8 Fab interaction interface. FIG. 62A illustrates the structure of GFRAL with the core 8D8 interaction interface amino acids (GFRAL epitope residues) on GFRAL highlighted in a space-filled surface model. FIG. 62B also illustrates the core interface amino acids on GFRAL covered by 8D8 in a space-filled surface model. FIG. 62C illustrates the structure of GFRAL in a ribbon diagram with boundary 8D8 Fab interaction interface amino acids on GFRAL (GFRAL epitope residues) highlighted in a space-filled surface model. FIG. 62D also illustrates the boundary interface amino acids on GFRAL covered by 8D8 in a space-filled surface model.

FIGS. 63A, 63B, 63C and 63D illustrate the core and boundary amino acid residues in the GFRAL/5F12 Fab interaction interface. FIG. 63A illustrates the structure of GFRAL with the core 5F12 interaction interface amino acids (GFRAL epitope residues) on GFRAL highlighted in a space-filled surface model. FIG. 63B also illustrates the core interface amino acids on GFRAL covered by 5F12 in a space-filled surface model. FIG. 63C illustrates the structure of GFRAL in a ribbon diagram with boundary 5F12 Fab interaction interface amino acids on GFRAL (GFRAL epitope residues) highlighted in a space-filled surface model. FIG. 63D also illustrates the boundary interface amino acids on GFRAL covered by 5F12 in a space-filled surface model.

GFRAL amino acids at the interface of the GFRAL/8D8 Fab complex are shown in Table 47. To compare the interaction interface amino acids in GFRAL/8D8 Fab and GFRAL/GDF15 complexes, Table 47 further lists the GFRAL amino acids at the interface of the GFRAL/GDF15 complex, which is also shown in Table 38. Table 47 illustrates that core interaction interface amino acid residues on GFRAL that bind to GDF15 overlaps with the core interaction interface amino acids in the GFRAL/8D8 Fab interaction.

The amino acid sequence of a full-length precursor human GFRAL protein is shown below (see also Example 12, part C):

GFRAL sequences SEQ ID NO: 1797         10         20         30         40 MIVFIFLAMG LSLENEYTSQ TNNCTYLREQ CLRDANGCKH         50         60         70         80 AWRVMEDACN DSDPGDPCKM RNSSYCNLSI QVLVESNFQF          90        100        110        120 KECLCTDDFY CTVNKLLGKK CINKSDNVKE DKFKWNLTTR        130        140        150        160 SHHGFKGMWS CLEVAEACVG DVVCNAQLAS YLKACSANGN        170        180        190        200 PCDLKQCQAA IRFFYQNIPF NIAQMLAFCD CAQSDIPCQQ        210        220        230        240 SKEALHSKTC AVNMVPPPTC LSVIRSCQND ELCRRHYRTF        250        260        270        280 QSKCWQRVTR KCHEDENCIS TLSKQDLTCS GSDDCKAAYI        290        300        310        320 DILGTVLQVQ CTCRTITQSE ESLCKIFQHM LHRKSCFNYP        330        340        350        360 TLSNVKGMAL YTRKHANKIT LTGFHSPFNG EVIYAAMCMT        370        380        390 VTCGILLLVM VKLRTSRISS KARDPSSIQI PGEL

TABLE 47 residues on GFRAL Residues on GFRAL that bind to that bind to GDF15 8D8 in in GFRAL/8D8 Fab structure Core interaction Core interaction Boundary interaction interface amino acids interface amino acids interface amino acids LEU132 Glu136 LEU132 ALA135 Ala137 GLU133 GLU136 Val139 VAL134 VAL139 Gly140 ALA135 GLY140 Asp141 GLU136 VAL142 Val142 ALA137 ASN145 Val143 CYS138 ALA146 Cys144 VAL139 LEU148 Asn145 GLY140 ALA149 Ala146 ASP141 LEU152 Gln147 VAL142 LYS153 Phe173 VAL143 ILE196 Asn177 CYS144 PRO197 Ile178 ASN145 GLN200 Pro179 ALA146 SER201 Asn181 GLN147 ALA204 Ile182 LEU148 LEU205 Met185 ALA149 SER150 TYR151 PHE174 TYR175 ALA169 ALA170 ILE171 ARG172 PHE173 GLN176 ASN177 ILE178 PRO179 PHE180 ASN181 ILE182 ALA183 GLN184 MET185 LEU186 ALA187 PHE188 CYS189 Core interaction interface amino acid residues on GFRAL that bind to both GDF15 and 8D8 Fab are shown in bold.

An analysis of the GFRAL epitope presented to 5F12 Fab in the 5F12 Fab::GFRAL::8D8 Fab complex crystal structure showed that 5F12's mechanism of action is that of a non-competitive GDF15 inhibitor and does not involve blocking GDF15-binding to GFRAL. Exemplary core interaction interface amino acids on the GFRAL protein and on the 5F12 Fab CDRs for the heavy and light chains are shown in FIG. 64.

GFRAL amino acids at the interface of the GFRAL/5F12 Fab complex are shown in Table 48. To compare the interaction interface amino acids in GFRAL/5F12 Fab and GFRAL/RET complexes, Table 48 further lists the GFRAL amino acids at the interface of the GFRAL/RET complex, which is also shown in Table 40A. Table 48 shows core interaction interface amino acid residues on GFRAL that bind to both RET and 5F12 Fab. Residues on GFRAL which are critical for GFRAL/5F12 interactions: 5F12 binding epitope on GFRAL reveals a non-competitive inhibitory mechanism of action of blocking GFRAL/RET interactions.

TABLE 48 residues on GFRAL that bind to RET in RET/GFRAL/GDF15 Model residues on GFRAL that bind to Core interaction 5F12 in 5F12/GFRAL structure interface amino acids Core interaction Boundary interaction on GFRAL interface amino acids interface amino acids Gln246 Arg234 CYS233 Arg247 Arg238 ARG234 Arg250 GLN241 ARG235 Lys251 Ser242 HIS236 Cys252 Lys243 TYR237 Asp255 Trp245 ARG238 Glu256 Gln246 THR239 Asn257 Thr249 PHE240 Cys258 Arg250 GLN241 Ile259 Lys251 SER242 Ser260 Cys252 LYS243 Thr261 His253 CYS244 Leu262 Asp255 TRP245 Thr297 Asn257 GLN246 Gln298 Cys258 ARG247 Ser299 Ser260 VAL248 Thr261 THR249 Leu262 ARG250 LYS251 CYS252 HIS253 GLU254 ASP255 GLU256 ASN257 CYS258 ILE259 SER260 THR261 LEU262 SER263 LYS264 ASP266 LEU267 THR268 SER272 ASP274 CYS275 ALA278 CYS269 SER270 SER302 LEU303 ILE306 HIS309 LEU311 MET310 SER315 CYS316 Core interaction interface amino acid residues on GFRAL that bind to both RET and 5F12 Fab are shown in bold.

The contents of all references described herein are hereby incorporated by reference.

Other embodiments are within the following claims.

Sequence Listing

The present specification is being filed with a computer readable form (GRF) copy of the Sequence Listing. The GRF entitled 13370-090-888_SEQ_LISTING.txt, which was created on Aug. 23, 2018, and is 968,136 bytes in size, is incorporated herein by reference in its entirety.

Lengthy table referenced here US20220073627A1-20220310-T00001 Please refer to the end of the specification for access instructions.

Lengthy table referenced here US20220073627A1-20220310-T00002 Please refer to the end of the specification for access instructions.

Lengthy table referenced here US20220073627A1-20220310-T00003 Please refer to the end of the specification for access instructions.

LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220073627A1). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

1.-106. (canceled)
 107. An antibody or antigen-binding fragment thereof that binds to a GDNF Family Receptor Alpha-Like (GFRAL) protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises a VH complementarity determining region (CDR)1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:7, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:8; (b) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:15, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:16; (c) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:1, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:2; (d) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:11, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:12; (e) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:21, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:22; (f) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:23, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:24; (g) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:25, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:26; (h) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:37, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:38; or (i) the VH comprises a VH CDR1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:39, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:40.
 108. A polynucleotide or polynucleotides encoding the antibody or antigen-binding fragment thereof of claim
 107. 109. A vector or vectors comprising the polynucleotide or polynucleotides of claim
 108. 110. An isolated cell comprising the polynucleotide or polynucleotides of claim
 108. 111. An isolated cell comprising the vector or vectors of claim
 109. 112. An isolated cell producing the antibody or antigen-binding fragment thereof of claim
 107. 113. A method of making an antibody or antigen-binding fragment thereof that binds to a GDNF Family Receptor Alpha-Like (GFRAL) protein, the method comprising: (a) culturing a cell comprising the polynucleotide or polynucleotides of claim 108 under conditions that promote expression of the antibody or antigen-binding fragment thereof; and (b) isolating the antibody or antigen-binding fragment thereof.
 114. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim
 107. 115. A method for treating involuntary body weight loss in a human subject, the method comprising administering to the human subject the antibody or antigen-binding fragment thereof of claim
 107. 116. A method for treating cachexia in a human subject, the method comprising administering to the human subject the antibody or antigen-binding fragment thereof of claim
 107. 117. A method for treating a GDF15-mediated disease or disorder in a human subject, the method comprising administering to the human subject the antibody or antigen-binding fragment thereof of claim
 107. 118. A method for treating a cancer in a human subject, the method comprising administering to the human subject the antibody or antigen-binding fragment thereof of claim 107, wherein the human subject has elevated levels of GDF15.
 119. An antibody or antigen-binding fragment thereof that binds to a GDNF Family Receptor Alpha-Like (GFRAL) protein, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a VH complementarity determining region (CDR)1, a VH CDR2, and a VH CDR3 from the amino acid sequence set forth in SEQ ID NO:3, and the VL comprises a VL CDR1, a VL CDR2, and a VL CDR3 from the amino acid sequence set forth in SEQ ID NO:4.
 120. A polynucleotide or polynucleotides encoding the antibody or antigen-binding fragment thereof of claim
 119. 121. A vector or vectors comprising the polynucleotide or polynucleotides of claim
 120. 122. An isolated cell comprising the polynucleotide or polynucleotides of claim
 120. 123. An isolated cell comprising the vector or vectors of claim
 121. 124. An isolated cell producing the antibody or antigen-binding fragment thereof of claim
 119. 125. A method of making an antibody or antigen-binding fragment thereof that binds to a GDNF Family Receptor Alpha-Like (GFRAL) protein, the method comprising: (a) culturing a cell comprising the polynucleotide or polynucleotides of claim 120 under conditions that promote expression of the antibody or antigen-binding fragment thereof; and (b) isolating the antibody or antigen-binding fragment thereof.
 126. A method for treating a GDF15-mediated disease or disorder in a human subject, the method comprising administering to the human subject the antibody or antigen-binding fragment thereof of claim
 119. 127. A method for treating a cancer in a human subject, the method comprising administering to the human subject an antibody or antigen-binding fragment thereof of claim
 119. 